international journal of innovative pharmaceutical ... · high-performance liquid chromatography...

REVIEW ARTICLE Vishal et.al / IJIPSR / 4 (5), 2016, 544-558

Department of Quality Assurance ISSN (online) 2347-2154

Available online : www.ijipsr.com May Issue 544

A REVIEW ON RECENT ADVANCEMENT OF LC-MS: ULTRA

HIGH PRESSURE LIQUID CHROMATOGRAPHY-MASS

SPECTROMETRY (UHPLC-MS) AND ITS APPLICATIONS

1Vishal Modi*, 2Akash Dubey, 3Parixit Prajapati, 1Tarashankar Basuri

1Head of Department, Department of Pharmaceutical Chemistry, SSR College of Pharmacy, sayli road, silvassa-396230, INDIA

2Department of Quality Assurance Techniques, SSR College of Pharmacy, sayli road, silvassa-396230, INDIA

3Department of Pharmaceutical Chemistry, SSR College of Pharmacy,

sayli road, silvassa-396230, INDIA

Corresponding Author:

Vishal Modi

Department of Pharmaceutical Chemistry,

SSR College of Pharmacy,

sayli road, silvassa-396230, INDIA

E-mail: [email protected]

Phone: 9824931330

International Journal of Innovative

Pharmaceutical Sciences and Research www.ijipsr.com

Abstract

Nowadays, pharmaceutical manufacturer trying to reduce the cost and shorten the time of drug development. Focus is placed on recent developments in high efficiency LC separations, sensitive electro spray ionization approaches, and the benefits of amalgamating Liquid

chromatography-mass spectrometer (LC-MS) based approaches. The new high throughput Ultra high pressure liquid chromatography-mass spectrometer (UHPLC-MS) and/or

UHPLC/MS/MS focuses on shortening the overall analysis time, with higher resolution, high speed, high sensitivity, and increase separation efficiency .Metabolic profiling for screening analysis and detection and quantifying of very potent drugs mainly illicit drugs and drugs of

abuse in whole blood is made easier by this technique. With the availability of sensitive mass selective instruments, such as tandem mass spectrophotometers and high resolution unwanted

substance with minimal sample preparation.

Keywords: UHPLC-MS, LC-MS, Electro spray ionization.

mailto:[email protected]




INTRODUCTION

High-performance liquid chromatography (HPLC) is one of the main analytical techniques used

for drug analysis. As such HPLC meets many important criteria for analysis but it is time-

consuming and uses a high amount of solvents compared to other analytical techniques. Ultra-

high performance liquid chromatography (UHPLC) has been proposed as an alternative to HPLC.

The use of UHPLC systems for potent molecule analysis has gained widespread acceptance

among researchers. A UHPLC System is a HPLC system innovated with regards to dead volume,

detector sampling rate, injection Performance and is able to tolerate application Pressures up to

1,500 bar. It is been improved in areas like chromatographic resolution, sensitivity of analysis etc.

It is therefore advantageous to use UHPLC instrumentation for increasing demand to provide

faster and selective screening techniques for an expanding range of drugs for forensic

methodology and workplace drug screening. Reduced analysis time provide for rapid release of

data, reduced costs per assay, and greater sample throughput overall.

Other important methodology exploit the high mass resolution capabilities of specially designed

mass spectrometers where these are not easily available, and where precursor and product ions

both are present ,so qualification and quantification of potent analytes is made easier by UHPLC-

MS [1].

PRINCIPLE

The basic principle of this chromatographic technique is governed by an formula known as Van

Dee meter equation which shows the relationship between plate height (HETP or column

efficiency) and linear velocity (flow rate). According to this equation, the increase in efficiency of

UHPLC technique is not possible without reducing particle size than those used in conventional

HPLC technique.

The Van Deemeter equation:

H = A + B/v + Cv,

Where, and A, B and C are constants and v is linear velocity.

Here, A represents the Eddy mixing which is independent of velocity. The value of A is lowest

when column particles are uniformly small.

B is the natural diffusion tendency of molecules or axial diffusion which is affected by flow rate.

This effect is diminished at high flow rates, so this term is divided by v.

C is the kinetic resistance to equilibrium in the separation process [2-6].




Fig. 1: diagram showing importance of HETP in column efficiency

INSTRUMENTATION

There has been great interest in approaches to rapid up and/or increase the resolving power of the

analytical separation process, particularly with the making of columns packed with porous sub-

2μm particles which is to be used in very high pressure conditions. Use of small particle size

results in higher plate numbers, as well as rapid separations.

These effects are due to;

i) The chromatographic efficiency, N, is directly proportional to the particle diameterand

ratio of column length, L/dp..

ii) The mobile phase linear velocity, u, is inversely proportional to the (dp) particle diameter.

Generally, the particle size reduction generates a high backpressure (more than 400 bar) which is

very difficult with conventional instrumentation. Therefore, to benefit from the full potential of

columns packed with small particles, it is smarter to work with a chromatographic system that

withstands pressures up to 1000 bar [7].

REQUIREMENTS FOR UHPLC EXPERIMENTS

PUMPING SYSTEM

For achieving high peak capacity separations requires a higher pressure range than that obtainable

in HPLC instrumentation. Across a 15 cm long column packed with 1.7 um particles the

calculated pressure drop at the optimum flow rate for maximum efficiency is about 15,000 psi.

Therefore a pump must be capable of delivering solvent reproducibly and smoothly at these

pressures, which can operate in both the gradient and isocratic separation modes. Up to 15,000-psi

pressure limit (about 1000 bar) can be achieved to take full advantage of the sub 2 um particles

[8,9].




SAMPLE INJECTION

Sample introduction in UHPLC is very important. Injection valves which are used conventionally

either manual or automated are not designed to work at extreme pressure. Protection of the

column from extreme pressure fluctuations, the process of injection must be pulse-free and the

swept volume of the device required being very low to reduce band spreading. Rapid injection

cycle time is required to fully capitalize on the speed afforded by UHPLC, which in turn need a

high sample capacity. Least carryover with low volume injections are also needed to increase

sensitivity. Direct injection approaches are also there for biological samples.10-11

SAMPLE MANAGER

With the help of high pressure of sample introduction low dispersion is maintained through the

injection process and diagnostics as well as self-monitoring can be facilitated by pressures

transducers in series. Variety of micro titer plate formats (mid height, deep well or vials) can also

be used in a thermostatically controlled environment. Using the particular sample organizer, the

sample manager can inject up to 22 micro titer plates. The column heater is also controlled by

sample manager. Column temperatures in a range up to 65°C can be maintained. A “pivot out”

design allows the column outlet to be placed in nearer proximity to the source inlet of an MS

detector to reduce sample dispersion.12

QUALITY OF MOBILE PHASE AND BUFFERS

Ultra high purity solvents signify directly to the sensitivity of the mass spectrometer that

reduces chromatographic interferences. It is important to verify the absence of insoluble

particles in the solvents and, for this purpose; many important rules for the mobile phase

preparation have to be followed in UHPLC.

Use only high grade organic solvents: methanol or acetonitrile (ideally filtered through a

0.22 μm membrane) – it is possible to obtain acetonitrile of UHPLC grade from several

suppliers (BIOSOLVE®, FISHER SCIENTIFIC®, JT BAKER® etc…)

Use of high quality salts for preparation of buffered mobile phases

The water must be ultra-pure and filtered through a 0.22 μm membrane (Milli-Q® system

of similar high quality water is recommended)

Benefit: Submicron filtration ensures minimal clogging of instrument, columns and check valves

Minimizes the leaching of metal cations (Na+ and K+).




Benefit: Low metal content in mobile phase solvents reduces formation of metal ion adducts. Be

careful from the microbiological growth (mainly when using phosphate buffer): Use only freshly

prepared mobile phases. Be vigilant with the cleaning of glassware and do not top-off the bottles.

The chromatographic system and columns should ideally be stored with pure organic solvents

(methanol or acetonitrile) to limit the microbiological growth [7].

UHPLC COLUMNS

Resolution can be increased in small 1.7 μm particle packed column because efficiency is better

with use of smaller particles. Sample requires a bonded phase for the separation of the

components that provides both retention and selectivity. Column length should be selected

according to the needed efficiency (in isocratic mode) or peak capacity (in gradient mode) [7].

List of different columns used in UHPLC

ACE EXCEL 2 C18

ZORBAX ECLIPSE 1.8 XDB C18

WATERS ACQUITY 1.7 BEH C18

PHENOMENEX KINETEX 1.7 C18

ACE EXCEL 2 C18-ARACE EXCEL 2 C18-PFP

High Efficiency UHPLC columns are available in 2μm particle size. Optimized UHPLC columns

utilize incredibly developed low dispersion hardware which enables high efficiency UHPLC

separations up to a maximum pressure limit up to 1,000 bar (15,000psi). UHPLC columns are

also available with C4, CN, AQ, C18, C8, Phenyl and Silica phases. C18 bonded phases are the

most popular phases for UHPLC because they provide high selectivity and good retention for a

wide variety of sample types. In addition, they are more rugged and reliable than other bonded

phases. C18 bonded phases mainly depends on hydrophobic interactions (and sometimes shape

selectivity) to achieve satisfactory separations. But, C18 phases offer a limited number of

separation mechanisms which may lead to less than optimum separations in some cases and

complete lack of resolution of important peak pairs. ACE columns have offered C18 bonded

phases with the advantage of „extra selectivity‟. C18-PFP and C18-AR bonded phases have

proved to be extremely powerful tools to use in leveraging this „extra selectivity‟ to obtain

separations that may not be possible with „standard‟ C18 bonded phases. The introduction of

different UHPLC columns means that chromatographers now have more options within their

reach to achieve great results with their UPLC and UHPLC instruments [13,14].




Fig. 2: diagram of column used in UHPLC

Advantages of UHPLC

• Increases sensitivity.

• Selectivity, sensitivity, and dynamic range of LC analysis drastically increased..

• Resolution performance is maintained.

• Enhance scope of Multi residue Methods.

• UHPLC‟s rapid resolving power quickly quantifies related and unrelated compounds.

• Quick analysis by using of a new separation material of very fine particle size.

• Operation cost is can be reduced.

• Solvent consumption in reduced.

• Real-time analysis in step with manufacturing processes can be delivered.

• Assures end-product quality and final release testing [5,6].

Disadvantages of UHPLC

Requires more maintenance and reduces the life of the columns due to increased pressure column

of this type. So far performance similar or even higher has been reported by using stationary

phases of size up to 2 μm without the adverse effects of high pressure [5,6].

MASS SPECTROMETRY INSTRUMENTATION

Operation of mass spectrometer is based on converting the analyte molecules to a charged

(ionized) state, with frequently analysis of the ions and any fragment ions that are obtained during

the ionization process, on the basis of their mass to charge ratio (m/z). Many different

technologies are available for both ionization and ion analysis, resulting in many different types

of mass spectrometers with different combinations of these two processes.

Fig. 3: various components of mass spectrometer




ION SOURCES

ELECTROSPRAY IONIZATION SOURCE

ESI was developed by Fenn into a robust ion source which is capable of interfacing with highly

optimized LC and demonstrated its application to a number of important classes of biological

molecules. ESI works significant with moderately polar moieties and is thus well suited to the

analysis of many metabolites, drugs and peptides. Pumping of a liquid sample through a metal

capillary maintained at 3 to 5 kv and nebulized at the tip of the capillary to form a charged

droplets which are very fine.

The capillary is usually off-axis from, or orthogonal to, the entrance to the mass spectrometer in

order to reduce contamination. The droplets are quickly evaporated by the help of dry nitrogen

and heat, and the transfer of residual charge to the analytes from the charged droplets takes place

[15].

Later the ionized analytes are transferred from the high vacuum to the mass spectrometer by

series of tiny apertures and focusing voltages. The subsequent ion optics and ion source can be

functioned to detect positive or negative ions, and switching between these two modes within an

analytical run can be performed. Under general conditions, ESI is known as “soft” ionization

source, meaning that relatively low energy is imparted to the analyte, and hence little

fragmentation occurs.

This has been used in LC-MS analysis to identify components with common structural features

e.g. Glycopeptides from glycan‟s can be fragmented in-source to give 204 m/z reporter ions. This

feature has been used to identify glycopeptides in tryptic digests of proteins in order to

characterize the structure of the glycan‟s.

While ESI is the most widely used ion source for biological molecules, neutral and low polarity

molecules like lipids may not be efficiently ionized by this method.

ATMOSPHERIC PRESSURE CHEMICAL IONIZATION SOURCE

As with ESI, in atmospheric pressure chemical ionization (APCI), liquid is pumped from a

capillary and nebulized at the tip. A corona discharge taking place near the tip of the capillary,

initially solvent molecules and ionizing gas present in the ion source.

Ions then react with the analyte and ionize it via charge transfer. The technique is useful for

thermally stable and small molecules which are not well ionized by ESI. The technique has also

been applied to fat soluble vitamins and lipids [16,17].




Fig. 4: schematic diagram of atmospheric pressure chemical ionization source

MASS ANALYSERS

QUADRUPOLE ANALYSERS

A set of four parallel metal rods are present in quadrupole analyser. A combination of varying

(radio frequency) and constant voltages allows the transmission of a narrow band of m/z values

along the axis of the rods. It is possible to scan across a range of m/z values, resulting in a mass

spectrum by varying the voltages with time. Most quadrupole analysers scan speeds up to 1000

m/z per sec or more are common and operate at <4000 m/z. They usually operate at unit mass

resolution meaning that the mass accuracy is better than 0.1 m/z. Alternative to scanning, the

quadrupole can be set to monitor with a specific m/z value, then set to monitor another m/z value,

and soon. This is achieved by stepping the voltages. This technique is important for improving the

detection limits of specific targeted analytes because more detector time can be reduced for

detecting specific ions in place of scanning across ions that are not produced by the analyte.

Stepping can be carried out within few milliseconds and a panel of m/z values can be stepped for

the detection of many analytes. Ions can be induced to undergo fragmentation by collisions with

an inert gas such as argon or nitrogen, a process known as collision induced dissociation.

Quadrupole is one type of collision cell that has been designed to maintain the low pressure of the

collision gas needed for dissociation and transmit most of the fragment ions which are produced.

Mass spectrometer configurations which are particularly useful are obtained by placing a collision

cell between two quadrupole mass analysers. This combination is known as triple quadrupole

mass spectrometer which is an example of tandem MS in which independently application of two

or more stages of mass analysis are achieved. The advantage of tandem MS is greatly increased

specifically over the analysis carried out by single stage mass analysis. For example, 25-hydroxy




vitamin D3 gives 401 m/z as major M+H+ ion during ESI that loses water at the time of collision

induced dissociation to give a major 383 m/z product ion.12 Highly specific detection of 25

hydroxy vitamin D3 is possible if the first and third quadrupoles are set to transmit ions of only

401 and 383 m/z respectively. This method is termed as single reaction monitoring and the

fragmentation is denoted 401>383 [19].

Fig. 5: schematic diagram of quadrapole mass analyser

TIME-OF-FLIGHT ANALYSERS

The Time-of-Flight (TOF) analyser works by accelerating ions through a high voltage. The time

needed to travel down a flight tube to reach the detector and velocity of ions, depends on their m/z

values. The output of the detector as in term of time can be converted into a mass spectrum if the

initial accelerating voltage is pulsed.

The TOF analyser can gain spectra extremely rapid with high sensitivity. It also has high mass

accuracy, through which molecular formulas of small molecules can be determined [19].

ION TRAP ANALYSERS

Ion trap analysers apply three hyperbolic electrodes to trap ions in a 3-D space using radio

frequency and static voltages. Ejection of ions sequentially from the trap on behalf of their m/z

values to create a mass spectrum takes place.

Alternatively, by the application of an exciting voltage, a specific ion can be isolated in the trap

while other ions are ejected. An inert gas can also be entered into the trap to induce

fragmentation. An important feature of these ion trap analysers is the ability to isolate and

fragment ions many times in succession before the final mass spectrum is obtained, resulting in

so-called msn capabilities [19,20].




VARIOUS APPLICATIONS OF UHPLC-MS:

TESTING AND QUANTIFICATION OF ILLICIT DRUGS IN URINE USING UHPLC

COUPLED TO ACCURATE MASS AXION 2 TOF MASS SPECTROMETER.

LC CONDITIONS:

LC System:

Pump: PerkinElmer Flexar™ FX-10 pump

Flow: 0.4 ml/min

Mobile phase A: Water containing with 0.1% formic acid

Mobile phase B: Acetonitrile containing with 0.1% formic acid

Injection volume: 4 μl in partial fill mode

Column used: PerkinElmer Brownlee™ SPP C-18, 2x50 mm, 2.7 μm at 25 °C

MS Conditions:

Mass spectrometer: PerkinElmer axion 2 TOF MS

Ionization source: PerkinElmer Ultra spray™ 2 (Dual ESI source)

Ionization mode: Positive

Spectral acquisition rate: 3 spectra/sec

Capillary exit voltage: 100 V

Trap pulse™ mode: 100-1000 m/z

Table 1: Testing and quantification of illicit drugs in urine

Sl.

No

.

Analyte Formula Monoisotopic

Mass [M+H]+

Observed

Mass

Mass

Error

(ppm) 1 Methamphetamine C10H15N 150.277 150.1273 -2.66

2 Amiodarone C25H29I2NO3 646.0310 646.0312 0.39 3 Amphetamine C19H13N 136.1121 1361113 -5.88

4 Codeine C18H21NO3 300.1594 300.1594 -0.07 5 Diazepam C16H13Cln2o 285.0789 285.0792 0.98

6 Doxepine C19H21NO 280.1696 280.1695 -0.36 7 Haloperidol C21H23clfno2 376.1474 376.1474 0.00

8 Morphine C17H19NO3 286.1438 286.1434 -1.40 9 Flurazepam C21H23clfn3o 388.1586 388.1593 1.80

10 Alprolazem C17H13cln4 309.092 309.0900 -0.65 11 Cocaine C17H21NO4 304.1543 304.1550 2.30

12 EDDP perchlorate

(fragment, [M-O4Cl]+) C20H24Clno4 278.1903 278.1906 1.08

13 Benzoyl ecognine C16H19NO4 290.1387 290.1394 2.41

14 Methadone C21H27NO 310.2165 310.2171 1.93 15 3,4 MDA C10H13NO2 180.1019 180.1016 -1.67

16 MDEA C12H17NO2 208.1332 208.1326 -2.88




17 MDMA C11H15NO2 194.1176 194.1169 -3.61 18 Phenyl propyl amine C9H13NO 152.1069 152.1060 -5.92

19 Oxycodone C18H21NO4 316.1543 316.1545 0.54

UHPLC-ESI-MS METHOD FOR THE QUANTIFICATION OF STEROID HORMONES.

EXPERIMENTAL PREPARATION OF STANDARD SOLUTION:

Steroid solution (single standard) of 1 mg/ml was prepared in 50:50 (v/v) water acetonitrile.

Method Parameters

Column: blue orchid 175-1.8 C18, 50 x 2 mm ID

Eluent A: Water+ 0.1 % Formic Acid

Eluent B:Acetonitrile + 0.1 % Formic Acid

Flow rate 0.4 ml/min

Injection volume 5 μl

Column temperature 40 °C

Run time3.0 min

MS Conditions:

Mass spectrometer : MSQ Plus single quadrapole mass spectrometer

Ionization mode : positive mode, ESI

Needle Voltage : 3.5 kv

Cone Voltage : 20 V

Probe temperature : 350 °C

MS Detection parameters

Mode Full scan mode, m/z 270 – 370

RESULT

For MS detection, the mass spectra for every compound obtained. The highest intensity fragment

is chosen for quantification. Fragment ions having lower intensities can be used as qualifiers in

real samples.

Table 2: Quantification of steroid hormones

Steroid Ionization Mass

[g/mol]

M/z Expected

[M+H]+

M/z

Found

Corticosterone ESI + 346.4 347 347

Cortisone ESI + 360.4 361 361

Deoxycorticosterone ESI + 330.5 331 331

Norgestrel ESI + 312.3 313 313

Progesterone ESI + 314.5 315 315

Testosterone ESI + 288.4 289 289




IDENTIFICATION OF CANNABINOIDS IN BAKED GOODS BY UHPLC/MS.

EXPERIMENTAL CONDITIONS:

Standard and Sample Preparation were mixed and diluted to up to 10 ppm to prepare a stock

solution with methanol.

Chromatographic Conditions:

Chromatographic analyses were performed using the Accela UHPLC system (Thermo Fisher

Scientific)

Column: Hypersil GOLD PFP (per fluorinated phenyl) 1.9 μm, 100 x 2.1 mm

Flow Rate: 1 ml/min

Mobile Phase: A: Water with 0.06 % acetic acid

B: Acetonitrile with 0.06% acetic acid

C: Methanol with 0.06% acetic acid

Column Temperature: 45 °C

Injection:

Syringe Speed: 8 μl/sec

Flush Speed: 100 μl/sec

Flush Volume: 400 μl

Wash Volume: 100 μl

Flush/Wash Source: Bottle with methanol

Mass Spectrometer Conditions

MS analysis was carried out on MSQ Plus single quadrapole LC/MS detector with Xcalibur 2.05

(Thermo Fisher Scientific).

The MS conditions were as given:

Ionization: ESI

Polarity: Positive

Probe Temperature: 500 °C

Cone Voltage: 90 V

Scan Mode: mass range of 50-500 m/z with full scan

ESI Voltage: 3.5 kv

Scan Time: 0.2 s H

The cannabinoids were detected by using full scans (50-500 m/z) of the single quadrupole mass

spectrometer, and the extracted ion chromatograms from m/z310.5-311.5 + 314.5-315.5




Table 3: Identification of cannabinoids in baked goods

COMPOUND

RT min

(STANDARD

COMPOUND)

RT

(BROWNIE)

Min

RT

(COOKIE)

Min

Product

ion (m/z)

Product

ion (m/z)

Brownie

Product

ion (m/z)

Cookie

Cannabidol 4.1 -- -- 315.31 -- --

THC 5.1 5.1 5.1 315.33 315.33 315.27 Cannabinol 5.4 -- -- 311.31 -- --

Using UHPLC/MS Cannabinoids in baked goods can be identified with minimal sample

preparation. The preparation time up to 10 min and run time 8 min make this a very efficient

analytical method.

QUALITATIVE AND QUANTITATIVE ANALYSIS OF UHPLC-MS

Determination of polyphenols

Determination of pesticides

Determination of primary aromatic amines

Determination of benzodiazepines

Determinations of aflatoxins

Determination of coumarin and chlorogenic acid.

Mycotoxin analysis.

CONCLUSION

UHPLC-ms give increased resolution, speed and sensitivity for liquid Chromatography-mass

spectrometry. The main advantage of UHPLC-ms is a reduction of analysis time, along with

reduced solvent consumption, high throughput analysis and reduction in cost of analysis. From

the literature survey it can be concluded that all categories of pharmaceutical drugs can be

analyzed by UHPLC-ms method within a very short period of time and with less solvent

consumption. UHPLC-ms technology is transforming lives and laboratories, creating new

opportunities for business profitability, and bringing new meaning to quality. The literature

survey shows that research on UHPLC-ms analysis, both, at national and international level have

been successfully done on all categories of drugs.

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