the effects of heme from hemolyzed matrix on the stability...

THE EFFECTS OF HEME FROM HEMOLYZED MATRIX ON THE STABILITY OF

OCTREOTIDE IN K2 EDTA PLASMA

William Arthur Altizer

A Thesis Submitted to the

University of North Carolina Wilmington in Partial Fulfillment

of the Requirements for a the degree of

Master of Science

Department of Chemistry and Biochemistry

University of North Carolina Wilmington

2012

Approved by

Advisory Committee

John Tyrell Scott Wright

James Reeves

Chair

Accepted by

_______________________

Dean, Graduate School

ii

TABLE OF CONTENTS

ABSTRACT ................................................................................................................................... iii

ACKNOWLEDGEMENTS ........................................................................................................... iv

LIST OF TABLES ...........................................................................................................................v

LIST OF FIGURES ....................................................................................................................... vi

INTRODUCTION ...........................................................................................................................1

METHODS ......................................................................................................................................6

RESULTS AND DISCUSSION ....................................................................................................30

CONCLUSIONS............................................................................................................................60

LITERATURE CITED ..................................................................................................................62

iii

ABSTRACT

An issue during blood collection for drug analysis is that the membranes of the red blood

cells can be damaged and leak their contents into the collected specimen. Hemoglobin, one of

the components of red blood cells, is comprised of four globular protein subunits and inside each

subunit is a protein chain tightly associated with a non-protein heme group. The free heme

compounds or free iron Fe (II) from the ruptured red blood cells could interact with other

components found in the collected specimen. This research was done to see if the drug

octreotide would be affected by the presence of hemolyzed matrix and in particular heme. The

results of this research showed that in the plasma treated with the anticoagulant dipotassium

ethylenediaminetetraacetate (K2 EDTA) octreotide was stable, for up to three hours, when

hemolyzed matrix or heme was added to a prepared sample. The research also looked at the

stability of octreotide in solutions containing certain metals. The metals aluminum, iron, and zinc

reduced the amount of recovered octreotide.

iv

ACKNOWLEDGEMENTS

I want to extend my thanks to my wife Tamara who has helped to keep me going during

the process of working towards my Masters Degree. To my parents and my son, Liam, thank

you for being so supportive during these past few years.

To Pharmaceutical Product Development, LLC (PPD) I want to say that I appreciate the

opportunity that they have opened up for their employees to give them the chance to further their

education. Also to Dr. Scott Wright who has been my mentor for the program at PPD I want to

extend my thanks for the encouragement and guidance.

To Dr. John Tyrell thank you for your support and encouragement during my time in the

program. I really appreciate your dedication to seeing that the students in the program are

successful in not only completing the program, but asking feedback so that that the program can

better serve future students.

I also want to thank the University of North Carolina Wilmington for putting forth a

distance education program for Master of Science degree in chemistry. Without this wonderful

opportunity I may never have found the time to continue my education.

v

LIST OF TABLES

Table Page

1. The high pressure liquid chromatography gradient program used for the analysis of octreotide

samples ...........................................................................................................................................23

2. The high pressure liquid chromatography gradient program used for the second octreotide

sample analysis method .................................................................................................................24

3. The standard electrode potentials of aluminum, copper, iron, and zinc ........................................28

4. A fourfold dilution of an octreotide sample in four different matrix combinations ......................33

5. A serial dilution of samples with octreotide and octreotide internal standard to eliminate

hemolyzed whole blood matrix effects on solid phase extraction. ..............................................34

6. Incubation of octreotide in matrix for 0 and 3 hours at room temperature and 37⁰C ....................36

7. Incubation of octreotide IS in matrix for 0 and 3 hours at room temperature and 37⁰C ...............37

8. Octreotide and octreotide internal standard samples were treated with hemin, heme, sodium

dithionite, Fe II or Fe III to show that octreotide and its internal standard are stable during a 1

hour room temperature incubation with these compounds ............................................................38

9. 10 ng/mL octreotide in 20:80 acetonitrile/water with 0.1% formic acid samples treated with

0.3mM aluminum, copper, iron, or zinc. Only the samples treated with zinc displayed a loss

of octreotide ...................................................................................................................................39

10. The 500ng/mL concentration of octreotide is converted to micromoles (µM) and compared to

the 300 µM concentration of the metals to show the ratio between octreotide and the metals. ....40

11. 10 ng/mL octreotide in 20:80 acetonitrile/water with 0.1% formic acid samples treated with

aluminum, copper, iron, or zinc after being stored in a refrigerator for 13 days ...........................54

12. Masses of the amino acids and metals involved in the research ....................................................59

vi

LIST OF FIGURES

Table Page

1. Structure of Octreotide .....................................................................................................................1

2. Structure of Somatostatin .................................................................................................................2

3. Picture of unhemolyzed matrix and three different levels of hemolysis .........................................3

4. Structure of Heme ............................................................................................................................4

5. Structure of Artemisinin ..................................................................................................................5

6. A diagram of a standard solid phase extraction cartridge. ...............................................................7

7. An example of solid phase extraction ............................................................................................10

8. Examples of analytical, narrow bore, and capillary HPLC columns .............................................11

9. The structure for the steroids Estradiol and Equilin-d ...................................................................13

10. Schematics of 4 basic pore types used in HPLC stationary phase .................................................14

11. A representation of an injector valve in the load, where the sample is place inside the loop.

When the valve switches to the inject position the sample is washed off of the loop into the

HPLC liquid stream .......................................................................................................................15

12. An example of a basic HPLC setup ...............................................................................................16

13. A representation of a single quadrupole mass spectrometer ..........................................................17

14. Quadrupole images, the left side image is looking down the barrel of the quadrupoles and

the right image is a side view of ion oscillating down the quadrupoles ........................................18

15. The schematic shows a simplified view of the LC/MS/MS analysis of a sample .........................19

16. A display of a mass spectrometry scan where the intensity is displayed on the y-axis as

counts per second (cps), and the mass-to-charge ratio (m/z) is displayed on the x-axis ...............20

17. A total ion HPLC-MS chromatogram where the intensity is displayed on the y-axis as counts

per second (cps), and the time is displayed on the x-axis in minutes ............................................21

18. Octreotide in plasma treated with hemolyzed whole blood at 3 different incubation times..........30

vii

19. Octreotide internal standard in plasma treated with hemolyzed whole blood at 3 different

incubation times. ............................................................................................................................31

20. Octreotide in plasma treated with non-hemolyzed whole blood at 3 different incubation times. .31

21. Octreotide internal standard in plasma treated with non-hemolyzed whole blood at 3 different

incubation times. ............................................................................................................................32

22. Q1 scan of the 500ng/mL octreotide control sample in 20:80 acetonitrile/water with 0.1%

formic acid .....................................................................................................................................40

23. Q1 scan of a 500ng/mL octreotide sample in 20:80 acetonitrile/water with 0.1% formic acid

treated with aluminum ...................................................................................................................41


treated with copper .........................................................................................................................42


treated with iron .............................................................................................................................43


treated with zinc .............................................................................................................................44

27. High pressure liquid chromatography analysis of the 500ng/mL octreotide control sample in

20:80 acetonitrile/water with 0.1% formic acid .............................................................................46

28. High pressure liquid chromatography analysis of a 500ng/mL octreotide sample in 20:80

acetonitrile/water with 0.1% formic acid treated with aluminum ..................................................47


acetonitrile/water with 0.1% formic acid treated with iron ...........................................................48


acetonitrile/water with 0.1% formic acid treated with zinc ...........................................................49

31. A scan of mass 410 to 620 from the time point of 1 to 1.6 minutes for the octreotide control

sample ............................................................................................................................................50

32. A scan of mass 410 to 620 from the time point of 1 to 1.6 minutes for octreotide treated with

iron .................................................................................................................................................51


zinc .................................................................................................................................................52


aluminum .......................................................................................................................................53

viii

35. A full scan from the time point of 0.85 to 1.1 minutes of the HPLC analysis of octreotide

treated with aluminum after a thirteen day incubation ..................................................................55


treated with iron after a thirteen day incubation ............................................................................56


treated with zinc after a thirteen day incubation ............................................................................57

38. Full scan from the time points of 0.86 to 1.22 minutes of the HPLC analysis of the octreotide

control sample after a thirteen day incubation ...............................................................................58

INTRODUCTION

Octreotide, H-D-Phe-Cyc-Phe-D-Trp-Lys-Thr-Cyc-Thr-ol, Figure 1, [1] is a synthetic

analog of the octapeptide hormone somatostatin [2] which is a growth hormone-inhibiting

hormone (GHIH). Somatostatin, Figure 2, regulates the endocrine system and affects

neurotransmission and cell proliferation by interacting with G-protein-coupled somatostatin

receptors and is involved in the release and inhibition of many secondary hormones. Octreotide

is a more potent inhibitor of insulin, growth hormones and glucagon than the naturally occurring

somatostatin. The salt form of octreotide, octreotide acetate, is the FDA approved form of this

peptide and is what is available commercially to patients.

Figure 1: Octreotide [3]

2

Figure 2: Somatostatin [4]

During clinical trials, blood specimens are drawn from patients at several time intervals

after the drug has been administered. The data from this analysis can then be used to determine

how quickly a drug enters the bloodstream and how long it takes the body to metabolize the drug

and remove it from the bloodstream. Once the specimens are drawn, they are centrifuged to

separate the red blood cells, platelets, serum and the plasma layer. Samples from the plasma

layer were used in this research. Sometimes when specimens are drawn they are mishandled

and the red blood cells are damaged prior to being centrifuged causing their cellular components

to leak out into the rest of the collected specimen. This process is called hemolysis [5] and some

of the causes in specimen mishandling are: blood being drawn too vigorously into a syringe, the

needle diameter is too restrictive for blood flow, or the specimen is shaken or vortexed too

vigorously prior to centrifugation. As shown in Figure 3, moderate or greater hemolysis can be

3

easily confirmed if the normally yellow colored plasma specimen has a pink or even reddish hue,

but even minor amounts of hemolysis that are not visible to the unaided eye will result in trace

increases in enzymes levels in samples. [6]

Figure 3: The left tube is an unhemolyzed specimen and the tubes on the right display an

increasingly higher percentage of hemolysis. [7]

This research was done to see if the peptide octreotide is affected by the presence of

hemolyzed matrix and in particular by the presence of heme, a component of hemoglobin.

Hemoglobin comprises about 35% of the total volume of the components within a red blood cell.

It is comprised of four globular protein subunits; each comprised of a protein chain tightly bound

to a non-protein heme molecule. Heme, Figure 4, is comprised of a single iron ion, Fe (II), at the

center of an organic heterocyclic ring called a porphyrin.

4

Figure 4: Heme [8]

When a cell membrane is ruptured, its contents leak out into the rest of the specimen and

can cause one of three issues:

1. The extra material escaping from the ruptured cells can change the overall concentration

of the sample by dilution [9]. If the compound of interest is found in higher

concentrations in plasma, then when the cell membrane is ruptured the sample will

become diluted and the analytical results will be lower. If the compound is found inside

the blood cells then the analytical results may be higher because when the cell membrane

is ruptured more of the drug will be available for analysis.

2. The released material from hemolysis can increase the absorbance in the short

wavelength range. Analytical results that rely on photometric measurements may have

an increase in the detected signal leading to erroneous data.

3. Some chemical reactions can be affected by the presence of the hemoglobin and in other

cases the actual chemical tests can be effected [10].

5

In a paper by Zhang and Gerhard [11], the authors noted that heme reduced the

endoperoxide bridge within artemisinin, Fig 5, and caused the activation of the drug. Heme

(Fe2+

protoporphyrin-IX) was more efficient in the reduction than inorganic iron or hemoglobin.

Because sulfur is a VIA element like oxygen and forms double bonds and hydrogen bonding in

the same manner, the reduction observed with the endoperoxide bridge could be possible with

disulfide bonds in other compounds. This research was designed to see if the disulfide bond in

octreotide would be affected like artemisinin’s endoperoxide bridge.

Figure 5: Artemisinin [12]

6

METHODS

Octreotide acetate was procured from Sequoia Research Products and the internal

standard (IS) octreotide [13

C6Phe3] was procured from Pharmaceutical Product Development.

HPLC grade methanol and acetonitrile were purchased from Burdick and Jackson. Ammonium

hydroxide (30%) and potassium hydroxide (86%) were purchased from J.T. Baker. Formic acid

and phosphoric acid were purchased from Mallinckrodt. The reagent water used was filtered by

the in house Millipore system. The Isolute-96 100mg C18 (EC) SPE plate was purchased from

Varian. An Acquity UPLC BEH C18 vanguard pre-column, 2.1mm x 5mm, 1.7 µm, product

number 186003975 and an Acquity UPLC BEH C18 vanguard pre-column, 2.1mm x 100mm,

1.7 µm (product number 186002352) were purchased from Waters. Iron powder (product

number 44890), zinc powder (product number 14409), aluminum powder (product number

214752), iron (II) chloride tetrahydrate (product number 220299), iron (III) chloride (product

number 157740), hemin (product number 51280), sodium dithionite (product number), and

protoporphyrin-IX (product number) were purchased from Sigma-Aldrich. The copper wire

(product number 31284) was purchased from Riedel-deHaën.

The HPLC equipment used was an Agilent 1200 series binary pump, a Shimadzu LC-

10AD VP series pump, and a 10 port cheminert 2 position selector valve from Valco

Instruments Company Incorporated, a hot sleeve-15L set at 50°C from Analytical Sales and

Services and an API 4000 series LC/MS/MS mass spectrometer from AB Sciex. The solid phase

extraction was automated using a Tomtec Quadra 96 Model 320 liquid-handling system.

The stock solutions were prepared at 100 µg/mL in 0.1:20:80 formic

acid/acetonitrile/water. The hemolyzed blood was made by taking the K2 EDTA whole blood

7

and freezing, thawing, vortexing, and then refreezing for three cycles to ensure that all of the cell

membranes had ruptured [7].

During the course of this research octreotide samples were prepared in matrix, human K2

EDTA plasma, and in other tests they were prepared in solution, 0.1:20:80 formic

acid/acetonitrile/water (v,v,v). The samples in matrix were prepared for mass spectrometer

analysis by separating the analyte of interest, octreotide, from the other components found in the

matrix. This separation was done by solid phase extraction [13], SPE, which is a process where

compounds suspended in a liquid are separated from the other compounds depending on their

chemical or physical properties. Each sample is extracted through an individual cartridge, Figure

6, which is made up of the reservoir, frits, sorbent bed, and luer tip. Depending on the desired

action a sorbent bed can be chosen to retain the analyte of interest and allow the unwanted

compounds to flow through, or to retain the unwanted compounds and allow the analyte of

interest to flow through the sorbent bed. In the case of this research, the chosen sorbent bed of

the cartridge, weak cation exchange, retained octreotide and allowed cleaning and then elution of

octreotide into a clean reservoir for high pressure liquid chromatography, HPLC, analysis.

Figure 6: A diagram of a standard solid phase extraction cartridge. [14]

8

There are three main types of sorbent beds [15], or stationary phases, to choose for

compound isolation and they are called: normal phase, reverse phase and ion exchange. Ion

exchange solid phase extraction is used for analytes that are charged when in solution and is

divided into four sub categories of stationary phase: strong anion, weak anion, strong cation and

weak cation. A weak cation exchange cartridge was the solid phase extraction type used for this

research.

Weak cation exchange (WCX) stationary phase is comprised of a carboxylic acid group

that is bonded to a silica surface and has a pKa of approximately 4.8 so it will be negatively

charged in a solution of at least 2 pH units above this pKa. The elution solvent for a weak cation

exchange stationary phase used to release the analyte from the stationary phase is at least 2 pH

units above the weak cation exchange pKa value. Strong cation exchange (SCX) stationary

phase is comprised of an aliphatic sulfonic acid group that is bonded to a silica surface and has a

pKa of less than 1. These are used to isolate strong cationic, pKa 14 or greater, or a weak

cationic, pKa of less than 12, compounds in matrix with a pH of a least 2 unit below the

analyte’s pKa. Elution from a strong cation exchange stationary phase is done with a solution

that has a pH of at least 2 units higher than the analytes pKa.

Strong anion exchange (SAX) stationary phase is comprised of an aliphatic quaternary

amine group that is bonded to a silica surface and has a pKa greater than 14 so that it is charged

at all pHs when in an aqueous solution. It is used to isolate weak anionic and strong ionic

compounds, and a solution with pH 2 units greater than the pKa is used as a matrix. Analytes

bound to these types of stationary phases are released by a solution at least 2 pH units below the

analytes pKa. Weak anion exchange (WAX) stationary phase is comprised of an aliphatic

9

aminopropyl group that has a pKa around 9.8. A solution of at least 2 pH units lower than the

pKa of the stationary phase is used, but the solution must also be 2 pH units above the analyte’s

pKa. Weak anion exchange is used to isolate strong and weak anions because they can be eluted

from the amine functional group by using a solution at least 2 pH units above the stationary

phase’s pKa.

Normal phase SPE uses a polar stationary phase to isolate a polar analyte suspended in a

mid to nonpolar matrix, like hexane or acetone. The retention mechanism is the interaction

between the polar groups of the analyte and the polar functional groups of the sorbent bed. The

analyte is eluted from the stationary phase by passing a solvent more polar than the original

matrix through the stationary phase.

Reverse phase SPE uses a nonpolar stationary phase to bind a mid to nonpolar analyte

suspended in a moderately polar or greater matrix. The retention mechanism is the interaction

between the carbon-hydrogen bonds of the analyte and the stationary phase. A nonpolar solvent

is used to elute the analyte from the stationary phase.

A typical solid phase extraction has four steps: conditioning, sample loading, washing

and elution, Figure 7. In the conditioning step the dry sorbent bed is treated by adding a solvent,

typically methanol, to wet the bed and penetrate the bonded phase within the sorbent bed. Then

either water, or a buffer close the composition of the sample is washed through the bed to

prepare the bonded phase. In the sample loading step, the sample is added to the reservoir and

allowed to slowly pass through the sorbent bed so that the analyte can interact with the material

and be retained. During this and all other steps the fluids are allowed to either pass through the

bed using gravity, centrifugation or by adding negative pressure to the cartridge using a vacuum

pump. The vacuum pump method was used for this research.

10

The washing step is done to remove impurities and other interfering material from the

sorbent bed without causing the analyte to be released. This washing step is typically done with

water or an organic compound that will not disrupt the interaction between the analyte and the

sorbent bed. Once washed the cartridge is moved so that the analyte may be eluted into a clean

container and the analyte is then washed from the cartridge with a solution that severs the

interaction between the analyte and the sorbent bed. Sometimes the elution solvent is not

compatible with the chosen HPLC column and the elution solvent must be removed, typically by

evaporation, and the dried sample reconstituted with a solvent more compatible for HPLC

analysis.

Figure 7: An example of solid phase extraction. The conditioning step wets the stationary phase

and prepares it for the loading phase where the analyte binds to the stationary phase. The

washing step removes impurities that might interfere with analysis after the analyte is removed

from the stationary phase during the elution step. [14]

High pressure liquid chromatography [16], HPLC, is a form of column chromatography

where a solvent is forced under high pressures of up to 6000 psi through a column that has been

11

packed with a stationary phase. By using higher pressures, smaller particle sizes can be used in

the stationary phase allowing for more interaction between the stationary phase and the

compounds suspended in a sample. The use of high pressure also allows for quicker elution

times and the compounds can be eluted in a much narrower band, increasing the sensitivity of the

assay.

There are several variants of chromatography available to separate analyte(s) from other

components within a sample following sample introduction. The chromatography variants are

normal phase, reverse phase, ion exchange, displacement, size-exclusion, and bioaffinity. The

choice of solvent, or mobile phase, and the packing material of the column determine what

variant of chromatography that is being used. The packing material and mechanisms of isolation

and elution for normal phase, reverse phase and ion exchange are similar to those used in solid

phase extraction.

Figure 8: Examples of analytical, narrow bore, and capillary HPLC columns. [17]

The other factors in high pressure liquid chromatography columns are the internal

diameter, length, particle size, pore size, temperature, and the amount of pressure applied to the

12

mobile phase. There are four categories of internal diameters, Figure 8, for high pressure liquid

chromatography columns: large, analytical, narrow-bore, and capillary. Large ID columns are

over 10 mm in diameter and are used for industrial applications. Analytical scale columns have

an internal diameter of 4.6 to 6 mm and narrow-bore columns average 1 to 2 mm in internal

diameter and are used for fluorescence and liquid chromatography-mass spectrometry analysis.

Capillary columns which are under 0.3 mm in diameter are made from fused silica capillaries

instead of stainless steel that is used in the other columns.

Column length is another factor when choosing a high pressure liquid chromatography

column because the extra amount of stationary phase allows for more interaction with the

analyte(s) being isolated. When dealing with a sample that has molecularly similar analytes the

extra amount of stationary phase can be critical. The steroids estradiol and the deuterated equilin

internal standard (equilin-d4), Figure 9, are good examples of this situation because they have the

same mass and the only differences between these two are that estradiol has an alcohol in the 17

position while equilin-d4 has a ketone at the 17 position, a double bond between the carbons at

the 7 and 8 position, and deuterium instead of hydrogen at four locations. Some assays require

the monitoring of the steroids estrone, estradiol, and equilin along with their deuterated internal

standards and this is when separation can become a critical. Separation of these compounds

requires high efficiency chromatography afforded by increased column length and/or reduced

particle size.

13

Figure 9: The structure for the steroids Estradiol and Equilin-d. [18]

The particle size of a column refers to the size of the silica beads that make up the

stationary phase. The smaller the particle size the greater the total surface area inside the column

which can improve separation, but the smaller particle size requires a greater amount of mobile

phase pressure to maintain good analyte separation across the stationary phase. Particle sizes

vary depending on the application the column is being used for, but 3 µm and 5 µm are the most

commonly used particles sizes. Columns with a particle size below 3 µm are used in ultra

performance liquid chromatography, UPLC. Columns and the pressure required for optimum

performance can reach up to 15000 psi in UPLC.

Many of the stationary phases used in high pressure liquid chromatography analysis are

porous [19], Figure 10, which means that the stationary phase has little pockets in their structure,

and these pores increase the available surface area inside the column. This increased surface

area allows for more interaction between the stationary phase and the analytes allowing for

greater separation.

14

Figure 10: Schematics of 4 basic pore types used in HPLC stationary phase; (a) totally porous,

(b) perfusion, (c) nonporous, and (d) superficially porous particles. [19]

The consistency of the pressure applied to the mobile phase by a pump is critical for the

reproducibility of a high pressure liquid chromatography assay. Many high pressure liquid

chromatography methods use more than one solution as their mobile phase and these solutions

are applied by a pump and mixed prior to entering the column. The mixture is changed slowly by

a gradient process to release the analyte from the stationary phase and allow the analyte to flow

downstream to the detector.

The efficiency of a high pressure liquid chromatography column can be improved by

raising its temperature [20] because at elevated temperatures the viscosity of liquids decrease and

the diffusion coefficient increases. The increased diffusion coefficient allows for better

interaction between the stationary phase and the analyte and can improve peak shape. This also

reduces the backpressure caused by the tightly packed stationary phase of the column which

decreases the stress on the HPLC pump, improving its efficiency.

15

Figure 11: A representation of an injector valve in the load, where the sample is place inside the

loop. When the valve switches to the inject position the sample is washed off of the loop into the

HPLC liquid stream. [21]

Using reverse-phase chromatography, the form used for this research project, the initial

composition of the mobile phase can be, for example, a mostly aqueous moderately polar solvent

that closely resembles the eluent from the solid phase extraction used to isolate the analyte from

the matrix. The sample is applied to the mobile phase stream by an injector, Figure 11, which is

a valve that allows the user to insert an aliquot of the sample into a section of tubing called a

loop when the injector is in the load position. The valve is actuated and the injector switches to

the inject position that moves the loop in line with the pump, Figure 12, and flushes the sample

downstream into the high pressure liquid chromatography column. Once the sample reaches the

column, the analyte interacts with the stationary phase and is held in place by van der Waals

forces of the carbon-hydrogen interaction. The composition of the mobile phase is slowly

changed to flush away unwanted material that has some affinity for the stationary phase, and

then the mobile phase reaches a sufficient nonpolar composition that the analyte is washed out of

the column and into the detector.

16

Figure 12: An example of a basic HPLC setup. [21]

The detector used was a quadrupole mass analyzer mass spectrometer [22] which is an

analytical instrument used to measure the mass-to-charge ratio (m/z) of charged particles. The

basic principle is to introduce a compound, which in case of the research is dissolved in a liquid

mobile phase, and ionize the chemical compounds to generate charged molecules and then

measure their mass-to-charge ratio. Once a sample is loaded into a mass spectrometer it

undergoes vaporization and the components are ionized. There are several forms of ionization

coupled to mass spectrometers, such as: electrospray ionization (ESI), atmospheric pressure

chemical ionization (APCI), fast atom bombardment (FAB), photoionization (PI), field

desorption (FD), direct analysis in real time, and secondary ion mass spectrometry. The one

used in the course of this research was electrospray ionization [23].

The ions are separated according to their mass-to-charge ratio by an electromagnetic field

generated by four circular rods, called quadrupoles, which are set in parallel to each other, Figure

17

13. The separation is done by stabilizing the trajectory of the selected mass-to-charge ratio in the

oscillating field generated by the quadrupoles, Figure 14. The opposing rods of the quadrupoles

are connected together electrically and a radio frequency is applied to each pair of the rods. Ions

that do not have the correct mass-to-charge ratio have an unstable trajectory and ultimately

collide with one of the rods and are deflected away from the detector located at the end of the

quadrupoles. The electrical potentials of the quadrupoles can be changed to allow a wide range

of mass-to-charge values to be monitored in either large groups or in succession.

Figure 13: A representation of a single quadrupole mass spectrometer. [24]

18

Figure 14: Quadrupole images, the left side image is looking down the barrel of the quadrupoles

and the right image is a side view of ion oscillating down the quadrupoles. [25]

The Sciex 4000 series mass spectrometer used for this research is a type of mass

spectrometer called a tandem mass spectrometer, or LC/MS/MS. A tandem mass spectrometer

[26], Figure 15, is capable of two rounds of mass spec isolation of particles separated by

molecular fragmentation. Three quadrupoles are aligned in a series and in the first quadrupole

(Q1) the analytes from the ion source are isolated prior to entering the second set of quadrupoles.

The second set of quadrupoles stabilizes the ions while they collide with a gas, in this case

nitrogen, causing them to fragment by collision-induced-dissociation (CID). In the third

quadrupole (Q3) the resulting fragments are sorted and the radio frequency is optimized so a

specific mass-to-charge ratio will reach the detector located just beyond the third quadrupole.

This allows for greater selectivity from a sample that may contain multiple compounds with the

19

same mass-to-charge ratio, but due to their structure they may produce different fragments.

Tandem mass spectrometry allows for much greater selectivity than the single quadrupole. Also,

researchers can deduce the structure of the initial compound by studying the fragments produced

in the collision cell. This is done by isolating the known compound mass in Q1 and after

fragmentation in the collision cell (Q2), Q3 is set to scan from the mass-to-charge ratio of the

parent compound and lower so that all the produced fragments can be found. The size and

intensities of these fragments can be then used to deduce the structure of the original compound.

Figure 15: The schematic shows a simplified view of the LC/MS/MS analysis of a sample.

http://www.thefullwiki.org/Lipidomics

At the end of the quadrupoles is a detector that records either the charge induced or the

current produced when an ion passes through or impacts the surface. These ions pass through an

electron multiplier called a Faraday cup to increase the signal received from the quadrupoles

because the amount of ions can be quite small. Once collected the signal response from the

detector can be displayed as intensity, or counts per second (cps), on the y-axis versus the mass-

to-charge ratio (m/z), on the x-axis, Figure 16. This type of spectrum only displays the total

amount of material with a specific mass-to-charge ratio in a sample, but cannot distinguish

20

multiple compounds of that ratio in the sample. Usually these types of scans do not involve

liquid chromatography prior to introduction into the mass spectrometer, but are done by a direct

infusion of a sample into the ion source.

Figure 16: A display of a mass spectrometry scan where the intensity is displayed on the y-axis

as counts per second (cps), and the mass-to-charge ratio (m/z) is displayed on the x-axis.

21

Figure 17: A total ion HPLC-MS chromatogram where the intensity is displayed on the y-axis as

counts per second (cps), and the time is displayed on the x-axis in minutes.

Figure 17 shows a display of a chromatogram in which high pressure liquid

chromatography separation is done to the sample prior to entering the ion source of the mass

spectrometer. The mass spectrometer is set to scan a specific mass or range of masses being

introduced into the ion source. The chromatogram displays the intensity (counts per second) of

what is scanned versus the duration of the scan. This allows for compounds that have the same

mass-to-charge ratio to be separated prior to entering the mass spectrometer and gives

confidence that the data represents the compound(s) intended for analysis.

22

Samples were analyzed using a procedure validated by PPD [27]. Into a 96-position,

2.0-mL, square-well, conical bottom, polypropylene plate, a 200 µL aliquot of sample in K2

EDTA human plasma was added and then into each well and 500 µL of phosphate buffer, pH 7.0

was added. Using a Tomtec automated solid phase extraction pipetting system an Isolute-96

100mg C18 (EC) SPE plate was conditioned three times with 300 µL of methanol, followed by

300 µL of reagent water, also administered three times. After each addition vacuum was applied

carefully so that the bed material did not completely dry out. The sample was then loaded onto

the SPE plate by transferring three 275 µL aliquots, and between each transfer a partial vacuum

was applied followed briefly by full vacuum.

The SPE plate was then washed three times with 300 µL of water, followed by three 200

µL treatments with 30:70 acetonitrile/water. A full vacuum was applied with each addition.

Next the sample was eluted from the SPE plate with three 300 µL additions of 2:8:90 by volume

29% ammonium hydroxide/water/methanol into a new 96-position, 2.0-mL, square-well, conical

bottom, polypropylene plate that was placed in the vacuum chamber. The eluent was evaporated

under a nitrogen stream at 45⁰C, and once dry the samples were reconstituted with 150 µL of

1:19:80 ammonium hydroxide/acetonitrile/water.

The HPLC separation was performed with an Xterra MS C18, 2.1 mm ID and 50 mm

length column using 0.5% ammonium hydroxide as mobile phase A and acetonitrile as mobile

phase B; see Table 1 for the chromatographic gradient conditions. The column was attached to a

Sciex 4000 series LC/MS/MS mass spectrometer set to positive mode electrospray ionization.

Mobile phase A, 0.5% ammonium hydroxide in water, and mobile phase B, acetonitrile, were

used for the chromatographic gradient.

23

Step

Total

Time

(min)

Flow

Rate

(µL/min)

Mobile

Phase

A (%)

Mobile

Phase

B (%)

0 0.0 300 70 30

1 0.5 300 70 30

2 1.5 300 20 80

3 2.5 300 20 80

4 2.6 300 10 90

5 3.2 300 10 90

6 3.4 300 70 30

7 5.5 300 70 30

Table 1: The high pressure liquid chromatography gradient program used for the analysis of

octreotide samples.

A second extraction procedure was used in the later experiments due to concerns with the

stability of the 2:8:90 ammonium hydroxide/water/methanol used as the elution solution for the

C18 SPE plate. Another chemist in the lab reported that she had noticed a difference in recovery

between SPE plates in a multi plate extraction session, and after some investigation it was

determined that the 2:8:90 ammonium hydroxide/water/methanol mixture could not be older than

a few hours between plate extractions or the amount recovered from the SPE plate would be

lower in the second plate. To eliminate possible bias when comparing different sample

extractions to each other, a newer extraction method that uses a WCX SPE plate with a 1%

trifluoroacetic acid (TFA) in 75:25 acetonitrile/water as the elution solution was used [28].

Into a 96-position, 2.0 mL, square-well, conical bottom, polypropylene plate, a 200 µL

aliquot of sample was added into each well, followed by 500 µL of phosphate buffer, pH 7.0 and

50 µL of 30 ng/mL internal standard working solution. Using a Tomtec automated extraction

solid phase extraction pipetting system, a WCX µElution SPE plate was conditioned with 200µL

of methanol, followed by 200 µL of reagent water. After each addition the vacuum was applied,

being careful not allow the bed material to completely dry out. The sample was then loaded onto

24

the SPE plate by transferring two 225 µL aliquots. Between each transfer a partial vacuum was

applied then the full vacuum was applied briefly.

The SPE plate was then washed twice with 200 µL with 5% ammonium hydroxide, and

then twice with 200 µL with 20:80 acetonitrile/water. A full vacuum was applied after each addition.

Next the sample was eluted from the SPE plate with 25 µL 1% TFA in 75:25 acetonitrile/water

two times into a new 96-position, 2.0 mL, square-well, conical bottom, polypropylene plate that

was placed in the vacuum chamber. The eluent was diluted with 150 µL of water through the

plate and the plate removed and the elution block was sealed and vortexed for 1 minute.

The HPLC separation was performed with an Acquity UPLC BEH C18, 2.1mm x

100mm, 1.7 µm pore size, and the column was heated to 50°C to improve efficiency. See Table 2

for the gradient program. Mobile phase A, 0.1% formic acid in water, and mobile phase B, 0.1%

formic acid in acetonitrile, were used for the chromatographic gradient. The flow from the

column was attached to a Sciex 4000 series LC/MS/MS mass spectrometer set to positive mode

electrospray ionization.

Table 2: The high pressure liquid chromatography gradient program used for the second

octreotide sample analysis method.

Samples were prepared at different concentrations of hemolyzed whole blood in plasma.

The control sample was 0% hemolyzed whole blood in plasma and had only octreotide and the

Step

Total

Time

(min)

Flow

Rate

(µL/min)

Mobile

Phase

A (%)

Mobile

Phase

B (%)

0 0.0 250 80 20

1 1 250 80 20

2 4.7 250 60 40

3 4.8 300 2 98

4 6 300 2 98

5 6.1 250 80 20

6 7.5 250 80 20

25

octreotide internal standard added. The 2% hemolyzed whole blood in plasma was used as an

example of mild hemolysis of a specimen and 20% hemolyzed whole blood in plasma was

prepared to represent the extremely hemolyzed sample. An 8% hemolyzed whole blood in

plasma was prepared to give a third data point for trending. These were compared to samples

with non-hemolyzed whole blood at the same concentrations.

Most of the experiments were done at two different temperature settings to see if room

temperature or 37°C, average human body temperature, would enhance or suppress any effects

hemolyzed blood would have on octreotide. Room temperature was chosen because most

samples are not immediately centrifuged to separate the plasma from the rest of the material in

whole blood and body temperature was used because most causes of hemolysis happen at the

time of collection and the specimens would still be warm when any effects would be caused by

the hemolyzed red blood cells. These samples at room temperature and 37°C were also

subdivided into incubation times to see if a 0 hour, or no incubation time, at these temperatures

would generate a different response than an incubation time of 3 hours. Reimers, McCann,

Cowan and Concannon [29] showed that peptides like insulin, which has disulfide bonds in its

structure, were affected by storage time, hemolysis and storage temperature but they only

reported results at room temperature and 4°C.

Another set of samples was prepared to test whether adding octreotide to the sample prior

to the introduction of hemolyzed matrix would have an effect on recovery. This was done to see

if octreotide had become protein bound in the plasma layer, protecting it from the effects of the

hemolyzed blood. Four sets of samples were prepared; 1) octreotide was added into a plasma

sample and then diluted fourfold with additional plasma, 2) octreotide was added into a plasma

sample diluted fourfold with hemolyzed whole blood, 3) octreotide was added to a mixture of

26

25% hemolyzed whole blood in plasma and the sample was diluted with additional 25%

hemolyzed whole blood in plasma, 4) octreotide was added into hemolyzed whole blood which

was then diluted fourfold with plasma. The prepared samples were allowed to equilibrate for

thirty minutes to being diluted.

The final extraction test was conducted by preparing a high concentration sample of

octreotide at 1000ng/mL in three different matrixes, K2 EDTA human plasma, 20% hemolyzed

whole blood in K2 EDTA human plasma, and 5% hemolyzed whole blood in K2 EDTA human

plasma. These were allowed to equilibrate for 1 hour at room temperature before a 0.100 mL

aliquot was taken and diluted to 1mL, a 10 fold dilution with K2 EDTA human plasma, which

was allowed to equilibrate at room temperature for 30 minutes. This sample was diluted again

10 fold with K2 EDTA human plasma and allowed to equilibrate for 30 minutes before aliquots

were taken and extracted in six replicates for the experiment.

Test samples of 10 ng/mL octreotide were prepared in K2 EDTA human plasma. Spiking

solutions of hemin were prepared at 2 mM and 4 mM in 0.05 N sodium hydroxide, also a spiking

solution of 4 mM sodium dithionite was prepared in 0.05 N sodium hydroxide. The sodium

hydroxide used for the hemin and sodium dithionite were degassed with helium prior to use and

the hemin and sodium dithionite powder was put in a 37°C vacuum oven. After an hour under

vacuum the chamber was filled with argon gas and the samples were quickly spiked with sodium

hydroxide. All of this was done to remove as much oxygen from the samples so that when the

sodium dithionite is added to the hemin it will reduce the hemin protein from an oxidized form

creating heme. Removing as much oxygen as possible from the sodium hydroxide prior to

preparing the sodium dithionite solution helped to ensure that the hemin was converted to heme.

Heme contains Fe2+

protoporphyrin-IX and it should be reactive to the disulfide bonds in

27

octreotide whereas the Fe3+

protoporphyrin-IX of hemin is in the highest oxidation for iron and is

not reactive. Also prepared were solutions of protoporphyrin IX (1.13 mg/mL), ferric chloride

(397.6 mg/mL) and ferrous chloride (324.4 mg/mL), each of these solutions were prepared at a

concentration 2mM in water. The amount of free iron found in a highly hemolyzed sample prior

to the sample being centrifuged is 860 ng/mL [6]. Because iron settles in the serum phase after

centrifugation, the amount of free iron in plasma after centrifugation is almost negligible except

in cases of rare disorders. A 1 mL aliquot of the 4 mM heme and a 1 mL of 4mM sodium

dithionite were mixed so that the sodium dithionite would convert the hemin to heme and make a

2mM (1.23 mg/mL) solution [30]. The concentration of 1.23 mg/mL is equivalent to 1%

hemolyzed blood in plasma. Six 4mL aliquots of the 10ng/mL octreotide were taken from the

pool and each was treated with one of the prepared solutions; heme, hemin, sodium dithionite,

protoporphyrin IX, ferric chloride and ferrous chloride. These solutions were allowed to

equilibrate for 1 hour at 37°C before aliquots were taken and extracted along with a control of 10

ng/mL octreotide in K2 EDTA plasma.

In an early experiment a stock solution of octreotide was found to have degraded within

two weeks of being prepared possibly because of the use of an aluminum weigh boat that was

left in the solution at the time of preparation. Upon investigation the 100 µg/mL octreotide stock

solution, which had been fresh in the first experiment, had degraded to such an extent that the

peak response was tenfold less than a freshly prepared stock. The aluminum weigh boat may

have caused a metal ion catalyzed reduction of octreotide. Erlandsson and Hällbrink (31)

observed that zinc broke the disulfide bonds in certain peptides and aluminum may be having a

similar effect on octreotide. A new stock solution, prepared using a glass weigh boat, was

28

compared to the solution that had been prepared with an aluminum weigh boat to confirm the

loss of octreotide and the new stock was used in the future experiments.

A test was done to determine if zinc II, aluminum III, copper I, copper II, or iron III

would have an effect on the recovery of octreotide in solution. A 0.49 µM intermediate solution

of octreotide in 20:80 acetonitrile/water with 0.1% formic acid was prepared and 2mL of the

intermediate was aliquoted into10mL vials. A 300 µM solution of each metal was prepared and

added to a vial. A second set of vials was prepared and to them was added 10uL of methyl

acrylate (MA), which will bind to any broken disulfide bonds preventing them from reforming.

Copper was chosen as a control metal since it has a positive standard electrode potential, Table 3,

and should not react with octreotide.

Table 3: The standard electrode potentials of aluminum, copper iron and zinc.

The solutions were vortexed and allowed to sit at room temperature for 1 hour before

being centrifuged at 4000rpm for 15 minutes. The samples were then infused directly into the

mass spectrometer and a Q1 scan was done with a scan window from 410 to 610 m/z, see figures

23-27, to see if there was any response loss at the 510 mass. Octreotide’s molecular mass is

1019.24 g/mol, but it is a dual charged molecule so the m/z (mass/charge) ratio is approximately

510 m/z.

A 0.250 mL aliquot was placed into a 96 well block and a 5uL injection was made and

the sample was separated by HPLC using an Acquity UPLC BEH C18, 1.7 µm pore size, 2.1

29

column inner diameter, and 100 cm long column. This was done to help chromatographically

separate degraded material resulting from the interaction of the metals with octreotide. The high

pressure liquid chromatography eluent was analyzed by a Q1 scan of the mass-to-charge ratio

range of 410 to 610 m/z.

The solutions were stored at 2-8˚C and were assayed again 13 days later to see if there

was any further degradation of octreotide caused by the presence of metal in the solution. The

scan window was narrowed to scan between the range of 450 to 560 m/z, see figures 28-35.

30

RESULTS AND DISCUSSION

Hemolyzed whole blood was added to plasma containing octreotide at different

percentages and another set of samples were created using non- hemolyzed matrix. The initial

results of comparing 0, 2, 8, and 20 % hemolyzed whole blood to the same percentages in non-

hemolyzed whole blood was promising because the samples with hemolyzed matrix showed a

definite trend in recovery loss as the percentage of hemolyzed matrix was increased, Figure 18

(octreotide), and Figure 19 (internal standard). The samples that had the same concentrations of

unhemolyzed whole blood did not show the same trend, Figure 20 (octreotide) and Figure 21

(internal standard). The incubation samples had nearly the same response in the hemolyzed and

non-hemolyzed samples except for the 0 % hemolyzed at the 3 hour incubation time which was

much higher and was determined to be a double spike of the spiking solution. The actual cause

of the recovery loss was not due to the degradation of octreotide by hemolyzed whole blood, but

likely due to the extra cellular material clogging the pores of the solid phase bed of the

extractions plates.

Figure 18: Octreotide in plasma treated with hemolyzed whole blood at 3 different incubation

times.

31

Figure 19: Octreotide internal standard in plasma treated with hemolyzed whole blood at 3

different incubation times.

Figure 20: Octreotide in plasma treated with non-hemolyzed whole blood at 3 different

incubation times.

32

Figure 21: Octreotide internal standard in plasma treated with non-hemolyzed whole blood at 3

different incubation times.

In the comparison of a fourfold dilution (dil4) of 100 ng/mL octreotide in K2 EDTA

human plasma samples that were diluted with hemolyzed blood did have a much lower recovery

than the sample prepared and diluted with plasma, Table 4. The results from where octreotide

was added to the samples containing hemolyzed whole blood were also lower than the samples

prepared completely in plasma, but not as low of a recovery as the plasma samples diluted with

hemolyzed blood, see Table 4. The expectation was that the plasma dil4 with hemolyzed and the

25% hemolyzed in plasma dil4 with additional 25% hemolyzed should have had the same

amount of recovery. Since the matrix to be added was freshly vortexed prior to addition, the

sample diluted with hemolyzed whole blood would have gotten more cellular material than the

sample with 25% hemolyzed whole blood in plasma. This additional material would have

competed with the binding sites on the solid phase bed more than the other samples. The internal

standard tracked very similarly to octreotide and the internal standard was not added until after

the 4% phosphoric acid had been added to the sample aliquots. If the hemolyzed matrix was

causing degradation of octreotide the reaction should have been stopped or at least reduced by

33

the addition of the 4% phosphoric acid, and the four different preparations should have had

internal standard responses that were more comparable. This reinforces the hypothesis that the

active sites on the stationary phase of the solid phase extraction cartridges were being blocked by

the material from the hemolyzed matrix.

Octreotide

Plasma

diluted with

plasma

(cps)

Plasma

diluted with

hemolyzed

blood (cps)

25%hemolyzed

diluted with

25%

hemolyzed

blood (cps)

Hemolyzed

blood

diluted with

plasma

(cps)

179003 45111 139738 148158

206237 70783 156629 150432

171956 56466 158523 145082

230050 53787 159712 150358

198194 52364 151775 134139

197188 67278 154440 142745

Mean 197105 57631 153469 145152

Std Dev 18842 8819 6669 5644

Octreotide

Internal

Standard

Plasma

diluted with

plasma

(cps)

Plasma

diluted with

hemolyzed

blood (cps)

25%hemolyzed

diluted with

25%

hemolyzed

blood (cps)

Hemolyzed

blood

diluted with

plasma

(cps)

85984 52612 71279 79482

99207 63309 79485 78430

80705 52757 77531 74869

112387 51070 81207 75031

93342 71712 80270 71180

94532 105900.1 79111 77145

Mean 94360 58292 78147 76023

Std Dev 10051 8004 3270 2732

Table 4: The fourfold dilution of samples containing octreotide and octreotide internal standard

in four different matrix combinations. The data point highlighted in red for the internal standard

sample of plasma diluted with hemolyzed blood was eliminated from the calculations.

34

In the test done where the samples were diluted tenfold twice to almost completely

eliminate any interference of the solid phase extraction plate by components from hemolyzed

whole blood there was no difference in the three sets of samples, Table 5. So all of the previous

results that showed a loss of recovery may have been caused by the competition of the SPE plate

binding sites between octreotide and the cellular components that were released when the whole

blood was hemolyzed. This reinforces the notion that hemolysis can have an effect on analysis,

but it does not seem to interfere with octreotide.

Octreotide

Control in

Plasma

(cps)

Octreotide

in 20%

Hemolyzed

Blood in

Plasma

(cps)

Octreotide

in 5%

Hemolyzed

Blood in

Plasma

(cps)

Octreotide

Internal

Standard

Control in

Plasma

(cps)

Octreotide

Internal

Standard in

20%

Hemolyzed

Blood in

Plasma

(cps)

Octreotide

Internal

Standard in

5%

Hemolyzed

Blood in

Plasma

(cps)

616631 633118 631952 332892 325804 311899

586831 569145 621343 313958 309688 314400

570409 569337 611432 294064 277338 308868

465233 652382 458663 227276 342586 329372

550797 549542 676180 282719 259484 313954

582153 577829 580176 297599 295712 290086

Mean 562009 591892 596624 291418 301769 311430

Std

Dev 52091 40929 74447 35953 30751 12645

Table 5: A serial dilution of samples with octreotide and octreotide internal standard to eliminate

hemolyzed whole blood matrix effects on solid phase extraction.

Samples prepared with 0%, 2%, 8% and 25% hemolyzed blood in K2 EDTA plasma

were tested at room temperature (RT) and 37°C. The two temperature settings were also

compared at two different incubations times; one was a 3 hour incubation of the samples and the

others was prepared at the two temperatures and extracted immediately, these samples were

called 0 hour. This was done to determine whether temperature and/or duration between

35

preparation and extraction would have an effect on the stability of octreotide, Table 6, and

octreotide internal standard, Table 7. The data shows that as the percentage of hemolyzed blood

increases there is a corresponding drop in the recovery of octreotide, and this is true in both the

RT and the 37⁰C samples. The 3 hour incubations for both 37⁰C and room temperature had a

higher amount of octreotide recovery.

The analysis to eliminate cellular components as a factor in the extraction was also done

to see if hemin, heme, ferrous chloride, or protoporphyrin IX would cause the degradation of

octreotide. Ferric chloride was added to the experiment as a control because that highly oxidized

state of iron should not react with octreotide. The results, Table 8, confirmed the data from the

multi-dilution test that octreotide seems to be stable in matrix when samples are treated with

hemolyzed whole blood. None of the reagents added and incubated for 1 hour caused a loss in

the recovery of octreotide. An experiment conducted by adding iron powder to octreotide in

20:80 acetonitrile/water with 0.1% formic acid was performed and when scanned by Q1 MS

analysis the immediate analysis of iron showed no loss of octreotide, but after a 13 day

incubation the iron treated sample did have a loss of octreotide. Additional testing with a longer

incubation time should be done.

36

Octreotide

25% Hemolyzed

Blood in Plasma (cps)

8% Hemolyzed


2% Hemolyzed


100% K2 EDTA Plasma (cps)

37°C 3Hour

Incubation

37°C 3Hour

Incubation

37°C 3Hour

Incubation

37°C 3Hour

Incubation

27649 38434 47949 65520

22995 37536 50184 67479

22748 40442 51950 68158

16394 38127 52944 57498

25228 38110 53519 64553

26495 35206 54298 63088

Mean 23585 37976 51807 64383

Std Dev 4011 1685 2365 3853

Octreotide

Room Temperature

3Hour Incubation

(cps)

Room Temperature

3Hour Incubation

(cps)

Room Temperature

3Hour Incubation

(cps)

Room Temperature

3Hour Incubation

(cps)

28734 38562 42141 58106

27557 38116 56442 61691

25402 38193 52385 65592

28100 35758 54054 52961

26190 40243 61309 57338

22796 40587 56651 57137

Mean 26463 38577 53830 58804

Std Dev 2176 1739 6473 4335

Octreotide 37°C

0 hour (cps) 37°C

0 hour (cps) 37°C

0 hour (cps) 37°C

0 hour (cps)

23743 27438 48131 41997

15345 27893 46104 51418

17725 32523 40530 49267

16892 31220 26626 48546

12393 36827 40649 51178

18839 31295 47452 45721

Mean 17490 31199 41582 48021

Std Dev 3793 3421 8043 3603

Octreotide

Room Temperature

0 hour (cps)

Room Temperature

0 hour (cps)

Room Temperature

0 hour (cps)

Room Temperature

0 hour (cps)

27632 24777 27964 48517

20298 29854 36913 49759

20771 35735 43214 46119

21615 32965 40636 52305

17318 33536 41467 48621

14761 36649 40369 45296

Mean 20399 32253 38427 48436

Std Dev 4364 4365 5524 2529

Table 6: The incubation of octreotide in matrix for 0 and 3 hours at room temperature and 37⁰C. The 3

hour incubation had higher recovery than those not incubated at 37⁰C.

37

Octreotide Internal

Standard

25%

Hemolyzed Blood in

Plasma (cps)

8% Hemolyzed Blood in

Plasma (cps)

2% Hemolyzed Blood in

Plasma (cps)

100% K2 EDTA Plasma

(cps)

37°C 3Hour

Incubation

37°C 3Hour

Incubation

37°C 3Hour

Incubation

37°C 3Hour

Incubation

17254 22724 28013 40684

14372 22922 31034 41030

13695 24615 31103 41664

9558 22244 31935 34507

15770 23594 32018 38630

15689 22300 32538 39506

Mean 14390 23066 31107 39337

Std Dev 2669 903 1621 2606

Octreotide

Internal

Standard

Room Temperature

3Hour

Incubation (cps)

Room Temperature

3Hour

Incubation (cps)

Room Temperature

3Hour

Incubation (cps)

Room Temperature

3Hour

Incubation (cps)

16983 23797 25198 35416

17646 23517 33975 36183

14487 22442 31293 39037

16058 21428 33204 32220

16071 25759 36476 34045

13703 25967 33598 35009

Mean 15825 23818 32291 35318

Std Dev 1488 1793 3852 2276

Octreotide Internal

Standard

37°C

0 hour (cps)

37°C

0 hour (cps)

37°C

0 hour (cps)

37°C

0 hour (cps)

14143 16594 28071 25372

9316 16238 28382 29934

10684 20678 24330 29222

10430 18914 16111 28517

7486 21045 23355 31696

11309 19254 28969 27113

Mean 10561 18787 24870 28642

Std Dev 2210 2011 4872 2208

Octreotide

Internal Standard

Room

Temperature 0 hour (cps)

Room


Room


Room


16719 14723 16855 28775

12396 18147 21632 29181

12981 21437 26907 28235

13185 19404 25149 31793

10357 20462 24742 30545

8938 21550 23238 27004

Mean 12429 19287 23087 29255

Std Dev 2675 2579 3537 1700

Table 7: The incubation of octreotide IS in matrix for 0 and 3 hours at room temperature and 37⁰C. The

3 hour incubation had higher recovery than those not incubated at 37⁰C.

38

Table 8: Octreotide and octreotide internal standard samples were treated with hemin, heme,

sodium dithionite, Fe II or Fe III to show that octreotide and its internal standard are stable

during a1 hour room temperature incubation with these compounds. All of the data is in counts

per second (cps).

In the analysis of a direct infusion into the mass spectrometer of octreotide treated with

aluminum, copper, iron or zinc, Table 9, there was no significant difference between the

octreotide control sample (Figure 23) and those containing aluminum (Figure 24), copper (Figure

25) and iron (Figure 26). Zinc (Figure 27) was the exception; there were no analyte peaks at the

510 mass. The samples treated with methyl acrylate (MA) had similar results to the same

samples that were not treated so their Q1 scans are not shown. The methyl acrylate treatment

was done to see if the disulfide bonds were broken and reforming the methyl acrylate would bind

and prevent the broken disulfide bonds from reforming. Because both the treated and untreated

39

sample had similar results this means that disulfide bonds are not being reformed after being

broken. The loss of octreotide reinforces the results described by Erlandsson and Hällbrink [31]

that the addition of metallic zinc to a sample containing a peptide with a disulfide bond causes a

recovery loss of the sample by the reduction of the disulfide bond.

Compound Peak Intensity

(cps)

Octreotide control 1.20x106

Octreotide treated with Copper 1.25 x106

Octreotide treated with Iron 1.15 x106

Octreotide treated with Zinc No visible Peak

Octreotide treated with Aluminum 1.33 x106

Octreotide control treated with Methyl Acrylate 1.40 x106

Octreotide treated with Copper and Methyl Acrylate 1.25 x106

Octreotide treated with Iron and Methyl Acrylate 1.25 x106

Octreotide treated with Zinc and Methyl Acrylate No visible Peak

Octreotide treated with Aluminum and Methyl Acrylate 1.46 x106

Table 9: 10 ng/mL octreotide in 20:80 acetonitrile/water with 0.1% formic acid samples treated

with 0.3mM aluminum, copper, iron, or zinc. Only the samples treated with zinc displayed a

loss of octreotide.

The concentration of 300 µM for the metals was selected to reproduce what was done by

Erlandsson and Hällbrink [27] with zinc. When converted, Table 10, from ng/mL to micromolar

octreotide is 600 times less than the concentration of the metals. A future experiment could be

done with lower concentrations of the metals to see what the minimal ratio of metal to octreotide

is needed to have complete loss of octreotide.

40

Metal

Atomic

Weight

(g/mol)

Amount

(mg)

Final

Volume

(mL)

Concentration

(µM)

Copper 63.55 0.0762 2 300

Iron 55.85 0.075 2 300

Zinc 65.38 0.0785 2 300

Aluminum 26.98 0.0324 2 300

Octreotide 1019.26 1.0009 2 0.491

Table 10: The 500ng/mL concentration of octreotide is converted to micromoles/L (µM) and

compared to the 300 µM concentration of the metals to show the ratio between octreotide and the

metals.

Figure 22: Q1 scan of the 500ng/mL octreotide control sample in 20:80 acetonitrile/water with

0.1% formic acid.

41

Figure 23: Q1 scan of a 500ng/mL octreotide sample in 20:80 acetonitrile/water with 0.1%

formic acid treated with aluminum.

42


formic acid treated with copper.

43


formic acid treated with iron.

44


formic acid treated with zinc.

When the samples were analyzed by high pressure liquid chromatography, octreotide

which has a column retention time of 3.61 minutes, the control sample’s (Figure 27) peak height

was comparable to the peak heights of the sample treated with copper and aluminum (Figure 28),

which means that copper and aluminum caused no degradation to octreotide during the short

incubation time. All three samples had peak height responses between 4.0e7 and 5.0e7. In the

sample treated with iron (Figure 29) the peak height was 1.15e6 and the sample treated with zinc

(Figure 30) had no discernable peak at the retention time of 3.61 minutes. In the iron and zinc

45

treated samples there was an additional peak that appeared in the window between 1 and 1.6

minutes that did not appear in the other three samples. The loss of octreotide in the sample

treated with zinc reinforces the results describe by Erlandsson and Hällbrink [31] that the

addition of metallic zinc to a sample containing a peptide with a disulfide bond causes the

reduction of the disulfide bond. In the full Q1 scan a large peak appears at 1 minute in the zinc

and iron samples that does not appear in the others; this peak is 9 times higher in zinc that it is in

the iron sample. This shows that there is some metal related degradation caused by zinc. The

methyl acrylate addition did not result in any more loss in response when compared to the

samples not treated with methyl acrylate, so the effect caused by the addition of zinc is breaking

the disulfide bonds in a manner that is not allowing them to reform.

46

Figure 27: High pressure liquid chromatography analysis of the 500ng/mL octreotide control

sample in 20:80 acetonitrile/water with 0.1% formic acid. The retention time of the octreotide

peak is 3.61 minutes.

47

Figure 28: High pressure liquid chromatography analysis of a 500ng/mL octreotide sample in

20:80 acetonitrile/water with 0.1% formic acid treated with aluminum. The retention time of the

octreotide peak is 3.61 minutes.

48


20:80 acetonitrile/water with 0.1% formic acid treated with iron. The retention time of the

octreotide peak off of the column is 3.60 minutes.

49


20:80 acetonitrile/water with 0.1% formic acid treated with zinc. There is not octreotide peak at

the expected retention time of 3.61 minutes. At 1.12 minutes is a peak not seen in the control

sample.

50

Figure 31: High pressure liquid chromatography analysis of the 500ng/mL octreotide control

sample. The upper panel is same one from Figure 27 and the lower panel is the scan of all

masses from 410 to 610 m/z within the time points of 1.02 to 1.61 minutes which are highlighted

in the upper panel. The peaks observed in the lower panel are nothing more than background

noise averaging 1.2e4 (cps) in height.

In the split screen of Figure 31, the upper window is the same one seen in Figure 27 and

the lower window is the scan of of all peaks between the masses of 410 and 610 m/z during this

1 to 1.6 minutes window. This scan of octreotide is a baseline to compare against the samples

treated with iron (Figure 32) and zinc (Figure 33) and the large peak observed in those samples.

In the iron treated sample the scan gave clusters of multiple charged species that are likely the

result of a breakdown of octreotide into smaller amino acid chains. This is plausible because no

51

other compounds besides octreotide, iron and the solvent 20:80 acetonitrile/water with 0.1%

formic acid were present. The zinc treated sample had a similar result but the clusters were

fewer than iron and had more intense peak heights, meaning there were more of those masses

present in the sample. Finally, aluminum (Figure 34) and copper had the same chromatogram as

the octreotide control, meaning that there had been no reaction with octreotide.

Figure 32: High pressure liquid chromatography analysis of a 500ng/mL octreotide sample

treated with iron. The upper panel is same one from Figure 29 and the lower panel is the scan all


in the upper panel. The peaks observed in the lower panel show several small clusters of masses.

52


treated with zinc. The upper panel is same one from Figure 30 and the lower panel is the scan all


in the upper panel. The peaks observed in the lower panel show a few clusters of possibly multi-

charged particles.

53


treated with aluminum. The upper panel is same one from Figure 28 and the lower panel is the

scan all masses from 410 to 610 m/z within the time points of 1.02 to 1.61 minutes which are

highlighted in the upper panel. There are no clusters of peaks observed in this sample.

After 13 days the samples were reanalyzed and the samples with copper showed only

minimal change when compared to the control sample. The samples with iron, which had

oxidized into a rusty slurry, zinc and aluminum all had no analyte response at the 510 mass,

Table 11.

54

(cps)

Control 1.60 x106

Copper 1.34 x106

Iron 0

Zinc 0

Aluminum 0

Control with MA 1.40 x106

Copper with MA 1.50 x106

Iron with MA 0

Zinc with MA 0

Table 11: LCMS chromatographic responses of the peak at 3.19 minutes of 10 ng/mL

octreotide in 20:80 acetonitrile/water with 0.1% formic acid samples treated with aluminum,

copper, iron, or zinc after being stored in a refrigerator for 13 days.

The aluminum treated sample (Figure 36) that had been stored in a refrigerator for 13

days did not have a peak at the retention time of 3.19 minutes, but did have a peak between the

time points of 0.5 to 1.16 minutes that was not present on day 1, see Figure 34. The retention

time difference between day 1 and day 13 were attributed to a new analytical column. When

examined the early peak did show some new masses that were not in the day 1 sample, but not

the same intensity as those in the iron (Figure 37) and zinc (Figure 38) treated samples when

compared to the octreotide control sample (Figure 39). It could be that when aluminum reacted

with the octreotide peptide it broke the molecule differently than iron or zinc, explaining the

different cluster masses. Mantyh, et al [32] found that the metals aluminum, iron and zinc

promoted the aggregation of the peptide β-Amyloid, a large peptide of 36 to 43 amino acids

commonly associated with Alzheimer’s disease; octreotide is only an 8 amino acid peptide.

55

Figure 35: High pressure liquid chromatography analysis of octreotide treated with aluminum

after a 13 day incubation. The upper panel is a Total ion count (TIC) Q1 scan and the lower

panel is the scan all masses from 460 to 560 m/z within the time points of 0.63 to 1.16 minutes

which are highlighted in the upper panel. There are no clusters of peaks observed in this sample.

There is no observed octreotide peak at 3.19 minutes in the upper panel.

56

Figure 36: High pressure liquid chromatography analysis of octreotide treated with iron after a

13 day incubation. The upper panel is a Total ion count (TIC) Q1 scan and the lower panel is the


highlighted in the upper panel. There are clusters of peaks observed in this sample. There is no

observed octreotide peak at 3.19 minutes in the upper panel.

57

Figure 37: High pressure liquid chromatography analysis of octreotide treated with zinc after a

13 day incubation. The upper panel is a Total ion count (TIC) Q1 scan and the lower panel is the


highlighted in the upper panel, and there are clusters of possible multi-charged particle observed

in this sample. There is no observed octreotide peak at 3.19 minutes in the upper panel.

58

Figure 38: High pressure liquid chromatography analysis of the octreotide control sample after

a 13 day incubation. The upper panel is a Total ion count (TIC) Q1 scan and the lower panel is

the scan all masses from 460 to 560 m/z within the time points of 0.86 to 1.22 minutes which are

highlighted in the upper panel and there are some peaks observed in this sample. There is an

octreotide peak at 3.19 minutes in the upper panel.

59

Abbreviation Name mass (g/mol)

Phe Phenylalanine 165.19

Cys Cysteine 121.16

Trp Tryptophan 204.23

Lys Lysine 146.19

Thr Threonine 119.12

Al Aluminum 26.98

Cu Copper 63.55

Fe Iron 55.85

Zn Zinc 65.38

Table 12: Masses of the amino acids and metals involved in the research.

60

CONCLUSIONS

The objective of this research was to determine if the presence of hemolyzed blood and

more specifically the released heme from hemolyzed blood, in a plasma sample preserved with

K2 EDTA would have a detrimental effect on the structural stability of the peptide octreotide.

The results indicate that octreotide is structurally stabile in the presence of both hemolyzed

matrix and heme for an exposure of up to 3 hours before the samples are put through a solid

phase extraction for analysis. The presence of hemolyzed matrix was found to have an adverse

effect on the recovery of octreotide by solid phase extraction unless pre-treatment was done to

the samples to mitigate the competitive binding of the active sites of stationary phase of the SPE

cartridges.

Octreotide stability was found to be effected by the presence of metals like aluminum,

iron, and zinc. It is possible that these metals are binding with octreotide and changing its mass

to charge ratio so that it not being found in the expected scanning window. Another possibility is

that the metals are causing a breakdown of the peptide structure creating smaller peptides that

have a different mass to charge ratio so that they would not be found in the expected scanning

window.

Further research regarding the effect of heme or hemolyzed matrix on the stability of

octreotide should consider the long term storage issue. Because we now know that when stored

in solution in a refrigerator for nearly two weeks there is no loss in octreotide, current tests could

be extended to look for long term loss of octreotide in matrix. These tests would be done in

refrigeration and -20°C, the standard storage temperature for octreotide in matrix. These tests

could also be run with heme, hemin, protoporphyrin, and Fe2+ to see if a longer duration of

storage and temperature would have an effect.

61

Another question for further research is to find out whether the metals are reducing

octreotide into smaller chains or forming different sized clusters that are capable of having

multiple charge states. It is likely that both are occurring and that the metals are breaking down

the peptide structure and/or binding with the resulting pieces to form new structures. This

breakdown and/or formation of clusters would explain the loss of octreotide and the formation of

different masses for each metal. Calculations do not match a simple disulfide breakage and

metal ion complex, so it is likely that other bonds are being broken and complexes are forming

with the available metal ions. Looking at the masses of the amino acids and metals involved,

Table 12, reinforces this explanation. The scan masses considered would have to be extended

from approximately 250 m/z to approximately 1200 m/z, the limit of the API4000.

Another option would be to check the samples every 24 hours after the introduction of

metal to put together a profile of degradation caused by the introduction of metal. These profiles

could be run at the storage temperature of 4°C, -20°C and -80°C to see if temperature would

slow down the degradation caused by the metals. The reaction with zinc was very rapid, but iron

and aluminum took hours or even days to completely break down octreotide. Also, the amount

of metal needed to cause the loss of octreotide needs further study. The molar amount of the

metals was over 600 times greater than octreotide. By varying the concentrations of the metal

we could determine if there is a minimal amount needed to cause a loss of octreotide, and if there

is a saturation level where more metal will not cause an increase in the rate of octreotide loss.

62

LITERATURE CITED

1. Brown, N.J., American Journal of Medical Science, 1990. Oct, 300 (4), pp 267-273.

2. Brazeua, P., Vale, W., Burgus, R., Ling, N., Butcher, M., Rivier, J., Guillemin, R.,

Science, 1973, Vol. 179, No. 4068, pp. 77-79.

3. Ghassemi, A. H., van Steenbergen, M. J., Barendregt, A., Talsma, H., Kok, R. J., van

Nostrum, C. F., Crommelin, D. J. A., Hennink, W. E. Pharmaceutical Research, 2012

January; 29(1): 110-120.

4. http://www.lookchem.com/cas-511/51110-01-1.html

5. Carroro, P., Servidio, G., Plebani, M., Clinical Chemistry, 2000, Vol. 46, No 2, pp. 306-

308.

6. Frank, J.J., Bermes, E.W., Bickel, M.J., Watkins, B.F., Clinical Chemistry, 1978, Vol. 24,

No. 11, pp. 1966-1970.

7. http://www.cdha.nshealth.ca/pathology-laboratory-medicine/clinical-chemistry/hemolysis

8. Voet, D., Voet, J. G., Biochemistry, 1990, John Wiley and Sons, Inc, pp 542,.

9. Caraway, W.T., American Journal of Clinical Pathology, 1962, 37, pp. 445-464.

10. Pai, S. H., Cry-Manthey, M., Clinical Chemistry, 1991, 22, No. 6, pp. 408-410.

11. Zhang, S., Gerhard, G.S., Biorganic and Medicinal Chemistry, 2008, 16, pp. 7853-7861.

12. Meshnick, S.R., Taylor, T.E., Kamchonwongpaisan, S., Microbiological Reviews, June

1996, pp. 301-315.

13. Simpson, N.J.K., Solid-Phase Extraction: Principles, Techniques, and Application, 2000,

Marcel Dekker, Inc.

14. http://www.biotage.com/DynPage.aspx?id=35833

15. Thurman, E.M., Mills, M.S., Solid-Phase Extraction: Principles and Practice, 1998,

Wiley-Interscience.

16. Snyder, L.R., Kirkland, J.J., Dolan, J.W., Introduction to Modern Liquid

Chromatography, John Wiley & Sons, New York, 2009.

17. http://www.analytical-sales.com/HPLC_Columns.html

63

18. Creegan, J.A., Hidy, B.J., PPD Method, LCMSC233 Version 1.00, 2002.

19. Majors, R.E., LCGC, 2004, Vol. 1, No. 1.

20. Antia, F.D., Horváth, C., Journal of Chromatography A, 1988, Vol. 435, pp. 1-15.

21. www.lcresources.com/resources

22. Chapman, J.R., Practical Organic Mass Spectrometry, 1986, John Wiley & Sons,

Chichester, England.

23. Fenn, J.B., Mann, M., Meng, C.K., Wong, S.F., Whitehouse, C.M., Science, 1989, Vol.

246, No. 4926, pp. 64-71.

24. http://www.files.chem.vt.edu/chem-ed/ms/quadrupo.html

25. http://www.analyticalspectroscopy.net/ap8-23.htm

26. Yost, R.A., Enke, C.G., Journal of the American Chemical Society, 1978, Vol. 100, No.

7, pp. 2274-2275.

27. Hidy, B.J., PPD Method, LCMSC296 Version 1.01, 2006.

28. Ismaiel, O.A., Zhang, T., Hidy, B.J., PPD Method, LCMSC296 Version 2.01, 2009.

29. Reimers, T.J., McCann, J.P., Cowan, R.G., Concannon, P.W., Proceedings of the Society

for Experimental Biology and Medicine, 1982, 170, pp. 509-516.

30. Di Iorio, E., Methods in Enzymology, 1981, Vol. 76, pp. 57-72.

31. Erlandsson, M., Hällbrink, M., International Journal of Peptide Research and

Therapeutics, 2005, Vol. 11, No. 4, pp. 261-265.

32. Mantyh, P.W., Ghilardi, J.R., Rogers, S., DeMaster, E., Allen, C.J., Stimson, E.R.,

Maggio, J.E., Journal of Neurochmeistry, 1993, Vol. 61, No. 3, pp. 1171-1174.

the effects of heme from hemolyzed matrix on the stability...

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