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Instruction Manual for the HPLC Analysis of

Catecholamines in Urine Order Number 6000

IVD

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Chromsystems Instruments & Chemicals GmbH is certified according to DIN EN ISO 9001, DIN EN ISO 13485 and ISO 13485 CMDR. Products are produced and put into circulation according to IVD guideline 98/79/EC. © This document is protected by copyright. All rights reserved. Chromsystems Instruments & Chemicals GmbH Am Haag 12 82166 Gräfelfing Germany

Phone: +49 89 18930-0 Fax: +49 89 18930-299 www.chromsystems.com

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Contents…………………………………………………Page

1 Ordering information....................................................................................... 4

2 Introduction .................................................................................................... 6

3 Theory of electrochemical detection .................................................................. 8

3.1 .... General principle .................................................................................... 8

3.2 .... Influence of the working potential on the electrochemical detection ............... 8

3.3 .... Optimisation of the working potential ........................................................ 9

4 HPLC system ................................................................................................. 10

4.1 .... Installation and start-up .......................................................................... 10

4.2 .... HPLC parameters .................................................................................. 11

4.3 .... HPLC column ........................................................................................ 11

4.4 .... Care and maintenance of the HPLC system .............................................. 12

4.4.1 ... Pump and tubing ....................................................................... 12

4.4.2 ... Working electrode ..................................................................... 12

4.4.3 ... Reference electrode ................................................................... 13

4.4.4 ... Avoiding pump pulsation ............................................................ 13

4.4.5 ... Shut-down ................................................................................ 13

5 Sample preparation ...................................................................................... 14

5.1 .... Collection and storage of urine specimens ............................................... 14

5.2 .... Reconstitution of the urine calibrator ........................................................ 14

5.3 .... Reconstitution of the controls ................................................................... 15

5.4 .... Sample preparation procedure ............................................................... 16

5.5 .... Sample preparation - overview ............................................................... 17

5.6 .... Stability of the prepared samples (eluates) ............................................... 17

6 Data acquisition and evaluation ..................................................................... 18

6.1 .... Calibration of the data analysis system .................................................... 18

6.2 .... Quantitative evaluation with internal standard .......................................... 18

7 Quality control .............................................................................................. 19

8 Reference ranges .......................................................................................... 19

9 Conversion factors ........................................................................................ 20

10 Storage and lifetime of the reagents ............................................................... 20

11 Waste disposal ............................................................................................. 20

12 Examples of chromatograms .......................................................................... 21

12.1 .. Chromatogram of an aqueous calibrator ................................................. 21

12.2 .. Chromatogram of a prepared urine sample ............................................. 21

13 Trouble shooting ........................................................................................... 22

14 Literature .................................................................................................... 24

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Contents…………………………………………………Page

Appendix I: Automated Sample Preparation with the Gilson® ASPEC™ ................ 26

Appendix II: Hazardous substance information ................................................... 29

Appendix III: Notes on manual calculation .......................................................... 30

Appendix IV: Validation .................................................................................... 31

Appendix V: Declaration of Conformity .............................................................. 32

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1 Ordering information Order no. Product 6000 HPLC Reagent Kit

Catecholamines in urine Contents for 100 analyses: Mobile Phase 1000 ml Calibration Standard 10 ml Internal Standard 10 ml Neutralisation Buffer 2 x 300 ml Elution Buffer 2 x 300 ml Sample Clean Up Columns 100 pcs.

Components available separately

5001 Mobile Phase 1000 ml 5002 Mobile Phase 10 x 1000 ml 6003 Calibration Standard 10 ml 6009 Urine Calibration Standard 5 x 10.0 ml (lyoph.) 6004 Internal Standard 10 ml 6055 Neutralisation Buffer 300 ml 6006 Elution Buffer 300 ml 6007 Sample Clean Up Columns 100 pcs. 6000/A1 HPLC Reagent Kit

Preparation with the Gilson® ASPEC™ Catecholamines in urine Inhalt für 100 Bestimmungen: Mobile Phase 1000 ml Urine Calibration Standard 10.0 ml (lyoph.) Internal Standard 6 ml Neutralisation Buffer 300 ml Elution Buffer 250 ml Sample Clean Up Columns with DEC caps 2 x 50 pcs.

6000/A5 HPLC Reagent Kit

Preparation with the Gilson® ASPEC™ Catecholamines in urine Inhalt für 500 Bestimmungen: Mobile Phase 3 x 1000 ml Urine Calibration Standard 5 x 10.0 ml (lyoph.) Internal Standard 30 ml Neutralisation Buffer 5 x 300 ml Elution Buffer 5 x 250 ml Sample Clean Up Columns with DEC caps 10 x 50 pcs.

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6000/A9 HPLC Reagent Kit Preparation with the Gilson® ASPEC™ Catecholamines in urine Inhalt für 1000 Bestimmungen: Mobile Phase 6 x 1000 ml Urine Calibration Standard 5 x 10.0 ml (lyoph.) Internal Standard 2 x 30 ml Neutralisation Buffer 3000 ml Elution Buffer 2500 ml Sample Clean Up Columns with DEC caps 20 x 50 pcs.

Components available separately for Gilson® ASPEC™

5001 Mobile Phase 1000 ml 6009/T Urine Calibration Standard 10 ml (lyoph.) 6009 Urine Calibration Standard 5 x 10.0 ml (lyoph.) 6004/A1 Internal Standard 6 ml 6004/A5 Internal Standard 30 ml 6008/A1 Neutralisation Buffer 300 ml 6008/A9 Neutralisation Buffer 3000 ml 6006/A9 Elution Buffer 2500 ml 6007/A Sample Clean Up Columns with DEC caps 50 pcs.

Accessories 6100 HPLC column (equilibrated with test chromatogram) 1 pc.

Chromsystems calibrators and controls for catecholamines in urine 6003 Calibration Standard 10 ml 6009 Urine Calibration Standard 5 x 10.0 ml (lyoph.) 0040 Endocrine Urine Control, normal range 10 x 8.0 ml (lyoph.) 0050 Endocrine Urine Control, pathological range 10 x 8.0 ml (lyoph.)

Accessories for electrochemical detectors 41203 Working electrode, activated and tested 1 pc. 41211 Reference electrode Ag/AgCl 1 pc. 41239 KCl solution, 3 mol/l 50 ml

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2 Introduction The catecholamines adrenaline, noradrenaline, and dopamine are synthesised from the amino acids L-phenylalanine or L-3,4-dihydroxyphenylalanine and play a central role in the organism as hormones and neurotransmitters (1).

Fig. 1 Metabolism of catecholamines and metanephrines The determination of plasma, urine, and tissue catecholamine-levels is of clinical importance in the diagnosis of pheochromocytoma and some other tumors of the nervous system (2–5). These diseases are characterised by a greatly enhanced production of catecholamines in the respective tissue, resulting in increased circulation and excretion of catecholamines in urine. The urinary and plasma concentrations of catecholamines and their metabolites may then be several times above the upper reference limits (6). Screening for catecholamine secreting tumors is preferably done by the quantitative determination of the 24 h excretion of catecholamines in urine. For tumor localisation or in pharmacological functional tests it is necessary to measure plasma levels (3, 7).

COMT

MAO

MAO

COMT

THL

MAO

COMT

COMT

MAO

MAO

MAO

COMT

PNMT

Homovanillic acid

Vanillylmandelic acid

L-ADC

Tyrosine

DBH

Metanephrine

Noradrenaline

Dopamine

3,4-Dihydroxymandelic acid

3,4-Dihydroxyphenylacetic acid

Normetanephrine

Adrenaline

3-Methoxytyramine

DOPA

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The quantitative determination of urinary and plasma catecholamines is not only useful in differential diagnosis of hypertension but also for the evaluation of a number of other clinical and pharmacological aspects. The concentrations of noradrenaline and adrenaline are indicative for the activity of the sympathetic nervous system (8,9), and are important parameters in congestive cardiac insufficiency, coronary heart disease, diabetes mellitus, arteriosclerosis, acute asthma, and others (10-18). Also for scientific questions in the area of stress research and in sports medicine catecholamine levels provide useful information (19-22). Intended use: The Chromsystems reagent kit Catecholamines in urine is an in vitro diagnostic device to be used in clinical laboratories for the quantitative detection of adrenaline, noradrenaline and dopamine in patient urine samples via HPLC (high performance liquid chromatography with electrochemical detection). It is intended as a monitoring test for patients with suspected catecholamine-secreting tumour. Principle of the reagent kit: This reagent kit is designed for the reliable HPLC determination of adrenaline, noradrenaline and dopamine in urine. Prior to chromatographic separation, the analytes are separated from the urine matrix by ion exchange. Sample preparation requires pH-adjustment of the diluted urine, transfer onto the Sample Clean Up Column and two washing steps. A selected HPLC column in combination with a mobile phase, optimised for this particular separation, allows confident and reliable chromatographic quantification. With the Chromsystems HPLC kit, one person can analyse up to 100 plasma samples per day. The analytical method gives rapid and reliable results and is therefore suitable for routine analysis.

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3 Theory of electrochemical detection

3.1 General principle The most common electrochemical measuring technique in liquid chromatography is amperometry with a constant working potential. Conventional amperometric detectors employ a three-electrode cell consisting of a working electrode, a reference electrode and a counter electrode. The potential needed for oxidation or reduction reactions (polarisation potential) is applied between reference electrode (usually Ag/AgCl) and the working electrode. The counter electrode serves to maintain the potential and to prevent current flow at the reference electrode. An electrochemically active substance passing through the detector cell will be oxidised or reduced. The oxidative or reductive transformation of this substance leads to a loss or gain of electrons; the resulting current is detected by the measuring instrument, amplified and displayed as the chromatographic signal. Since only a limited number of functional groups and chemical structures are susceptible to redox processes at a particular working potential, electrochemical detection is not only characterised by high sensitivity but also by high selectivity. For the detection of catecholamines applies:

3.2 Influence of the working potential on the electrochemical detection Selection of the appropriate working potential is extremely important for the selectivity of the analysis. This means, ideally, choosing a working potential that yields the maximum detector signal for the desired substance, but at which none of the possible interfering substances (substances normally also present in the sample matrix) are detected. The relationship between the detector signal and the potential at the working electrode is shown in figure 2. Fig. 2 Correlation between working potential and detector signal

ROH

OH

- 2 e-

- 2 H+RO

O

Signal (nA or peak height/ peak area)

P1 P2 P3 = Popt.

Working potential [mV]

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At a potential less than P1 insufficient energy is available for electrochemical transformation of the molecules at the surface of the working electrode. Raising the potential to P2 increases the energy available, so that already a substantial proportion of the molecules reaching the working electrode will be converted. Raising the potential further, to P3, delivers sufficient energy to transform all molecules reaching the working electrode. No further increase in signal is achieved by a higher potential. The signal is now dependent only on the concentration of the substance (diffusion controlled plateau). Since no further increase in signal is possible after reaching the plateau, it is unnecessary to measure at potentials greater than P3. Indeed, at higher potentials selectivity will be lost, because the higher the available energy, the greater the number of substances that will be electrochemically transformed.

3.3 Optimisation of the working potential Experience has shown that the signal/working potential curve (see section 3.2) for a given substance differs from detector to detector. This is due to the fact that the reference systems (reference electrodes) in the different detectors are not completely identical and thus yield different reference potentials. This means, that the optimal working potential for an HPLC system with electrochemical detection must be determined empirically by injecting a standard mixture at different working potentials. For optimisation of catecholamine analysis the peak area or peak height of the internal standard dihydroxybenzylamine (DHBA) in the aqueous calibrator (order no. 6003) is used, since its electrochemical transformation requires the highest working potential. The following simple procedure is recommended (see fig. 3): 1. Set working potential to 360 mV and inject calibrator (6003). Determine peak area or peak height for DHBA. 2. Increase working potential by 40 mV. Inject calibrator (6003). Determine peak area or peak height for DHBA. If peak area/height increases by more than 15 %, then repeat step 2. If peak area/height does not change significantly, then reduce the potential by 40 mV. The optimal working potential determined by this method is generally +400 to +500 mV (vs. Ag/AgCl reference electrode) and is not influenced by routine maintenance of the measurement cell (e.g. replacing the KCl solution, activation of the working electrode). However, after major disturbance of the measurement cell (e.g. replacing the reference electrode) the optimisation of the working potential should be repeated.

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Fig. 3 Optimisation of the working potential

4 HPLC system

Caution: When using the reagents, please note the recommended hazards information in Appendix II.

4.1 Installation and start-up Connect the analytical column in the correct flow direction to the injector. Allow approximately 20 ml of the mobile phase to flow through the column at 1 ml/min. Then mount the capillary from the column outlet to the detector cell inlet. It is not necessary to degas the mobile phase before use. When the system is equilibrated, and the background current in the detection system is less than 5 nA the mobile phase can be recirculated (via an outlet capillary from the detector cell outlet back to the mobile phase bottle). As needle rinsing solution for the injector a mixture of ultrapure water (HPLC grade) with 5 % methanol is recommended. Detailed information for start-up of the single components of your HPLC system will be found in the instrument instruction manuals.

360 400 440 480

Working potential [mV]

NA = noradrenaline, A = adrenaline, IS = internal standard, DA = dopamine

Signal (Peak height/ peak area)

3rd injection

4th injection

2nd injection

NA A IS DA

1st injection

NA A IS DA

NA A IS DA NA A IS DA

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4.2 HPLC parameters Detection: Choose a working potential for electrochemical detection of catecholamines corresponding to the determined plateau value (see section 3.2). For detection of catecholamines and DHBA (internal standard) this will generally be 400–500 mV (against Ag/AgCl as reference electrode). After equilibration the background current in the detection system should not exceed 3 nA. Chromatographic separation: 1. Adjust the flow rate to 0.8–1.3 ml/min. The column backpressure should not exceed 200 bar). 2. The separation can be carried out at room temperature, but the temperature should be as constant as possible. Variations of temperature (and consequent variations in retention times) may be avoided by the use of a column oven. It is not necessary to degas the mobile phase before use. When the system is equilibrated, the mobile phase can be recirculated. Note: Degassing the mobile phase reduces the organic content of the eluent. This changes the chromatographic separation of the catecholamines and leads to marked increases in retention times. Retention times:

Analyte Retention time

(flow rate: 1 ml/min)

Noradrenaline approx. 4.5 min

Adrenaline approx. 5.5 min

Internal standard (DHBA) approx. 8.3 min

Dopamine approx. 13.5 min

Total analysis time is about 15 minutes. Variation in retention times: ±10 % Checking the separation efficiency To monitor the separation efficiency of the system a test analysis is recommended before analysing urine samples. For this purpose an aliquot of the calibrator is repeatedly injected. You can set the integration parameters (e.g. peak start and end markers) correctly on the basis of the chromatograms obtained. The last test injection can be used for calibration.

4.3 HPLC column The HPLC column for the analysis of catecholamines is supplied equilibrated and tested, and is ready to use. It must not be rinsed with any other solutions. The backpressure of a new column at a flow rate of 1.0 ml/min is about 75 bar (1100 psi) and may increase with column age and/or use. As long as the separations are satisfactory, a raised backpressure is of no consequence, but it should not exceed 200 bar (3000 psi).

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4.4 Care and maintenance of the HPLC system

4.4.1 Pump and tubing Analytical work in the high sensitivity range necessary for these analyses requires the highest cleanliness of all components. Therefore use only reagents and solvents of the appropriate degree of purity. Especially with electrochemical detection, even the slightest traces of electrochemically active substances lead to substantial increases in background or additional peaks. In most cases problems with electrochemical detection result from contamination of the HPLC system. As a rule a "preoxidation" (= passivation) of the HPLC system with nitric acid is recommended every 3–4 months (depending on the sample throughput). Passivation procedure: Before passivation, the HPLC column and the electrochemical detector must be disconnected! You should also passivate the capillary leading from the analytical column to the detector cell; simply integrate it into the capillary system with a union. Before rinsing with nitric acid, the mobile phase must be washed out with ca. 20 ml of ultrapure water (HPLC grade) at a flow rate of 1.5 ml/min. Then 15–20 % nitric acid is pumped through the capillary and injection system at a flow rate of 1.5 ml/min for 20 min. The injection unit should carry out the injection procedure several times: With an autosampler load 15–20 % nitric acid into the sample vials and inject a volume as large as possible. When using a manual injection system, switch from LOAD to INJECT at least 10 times. After passivation wash the nitric acid out of the system with ultrapure water (HPLC grade), again activating the injection procedure several times. The pH of the effluent rinsing water must reach the pH of the inflowing water before re-equilibrating the system again with the mobile phase.

4.4.2 Working electrode Prolonged use of the electrochemical detector may result in contamination of the active surface of the working electrode, leading to a decrease in sensitivity. To re-establish the sensitivity of a glassy-carbon working electrode treatment with chromosulphuric (chromic) acid is recommended.

Caution! Wear safety goggles and safety gloves and be extremely careful in handling chromosulphuric acid. Ensure that the electrode is completely dry before starting with the cleaning procedure.

Procedure: 1. Remove the working electrode from the analytical cell. Be careful with the electrode.

Touch it at the edges only. 2. Place the electrode on a flat surface. 1. Place a drop of chromosulphuric acid on the surface of the electrode. Moisten only the glassy-carbon

electrode surface (black centre). 3. Wait 2–5 minutes. 4. Rinse the electrode thoroughly with ultrapure water (HPLC grade). 5. Replace the electrode in the analytical cell. The active side points to the interior of the cell. 6. Re-equilibrate the system.

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4.4.3 Reference electrode (Only for electrochemical detectors from Chromsystems)

With time, the ions from the electrolyte solution (3 mol/l KCl) diffuse through the diaphragm into the mobile phase, thus shifting the potential of the reference electrode to higher values. To retain a defined potential in the reference system, it is essential to refill the reference electrode at least once a week with 3 M KCl solution. Be sure not to leave any air bubbles in the reference electrode! The white Teflon adapter fixes the reference electrode in the analytical cell. A porous glass diaphragm at the bottom of the adapter controls the diffusion of ions between mobile phase and reference electrode. Salts crystallising inside the cell can cause the diaphragm to crack, resulting in a leak between the eluent flow and the inner side of the reference electrode. If the Ag/AgCl-electrode has been in use for a longer time, AgCl-crystals can precipitate as a dark layer on the diaphragm. Such an AgCl precipitate on the diaphragm disturbs the balance of the reference system. The set working potential cannot be kept constant. To overcome this either replace the electrode adapter or try to clean the diaphragm with 25 % ammonia (place the Teflon adapter in 25 % ammonia over night, then rinse thoroughly with water).

4.4.4 Avoiding pump pulsation Electrochemical detection is based on an electrochemical reaction of the analytes at the surface of the working electrode. The reaction rate strongly depends on the speed by which the analytes are transported to the electrode. Pump pulsations cause discontinuous flow of the eluent through the flow cell, leading to an irregular reaction rate of the analytes at the electrode surface, and, therefore, to a very unstable baseline. For high sensitivity measurement it is recommended to use a pulse dampener.

4.4.5 Shut-down For an analysis-free period of up to one week it is recommended that the installed and equilibrated HPLC system be left running. The mobile phase can be recirculated at a reduced flow (0.2 ml/min). For longer periods the shut down procedure is as follows: 1. Disconnect and seal the HPLC column carefully. Do not rinse or add any preservative to the column.

Store the column in the mobile phase at room temperature. 2. Switch the electrochemical detector to "stand-by". Rinse the HPLC system with approximately 50 ml

water:methanol (50:50) 3. Remove the reference and working electrodes from the detector, rinse with water and store dry until

further use. Mark the active side of the working electrode.

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5 Sample preparation

Caution: When using the reagents, please note the recommended hazards information in Appendix II.

The samples can also be prepared automated with Gilson® ASPEC™. Please refer to Appendix I.

5.1 Collection and storage of urine specimens Normally 24 h urines are used for analysis. If this is very difficult or not possible, spontaneous urine samples can also be analysed. The data in this case should be referred to urine creatinine. The 24 h urine is collected in a suitable container containing 10 ml 25 % HCl. Urine treated this way is stable for at least 5 days at +2 to +8 °C. For longer storage aliquots of the 24 h urine should be frozen below –18 °C.

Note: It is the responsibility of the individual laboratory to use all available references and/or its own studies to determine specific stability criteria for its laboratory.

5.2 Reconstitution of the urine calibrator The calibrator (order No. 6009) is traceable to reference substances purchased from a certified supplier. After reconstitution, the calibrator is subjected to the entire sample preparation, analogous to patient specimens. The calibrator, so prepared, is used to calibrate the HPLC system. To reconstitute the lyophilised urine calibrator, pipette exactly 10.0 ml distilled water into the vial. Let the vial stand at room temperature for about 10–15 min, shake occasionally and gently until the vial contents are completely dissolved. Avoid exposure to direct sunlight! The actual concentrations depend on the batch and will be found on the information leaflet accompanying the calibrator.

Caution: This product has been manufactured from pooled human urine. Each donor contributing to this product is constantly subjected to medical control and judged as free of infectious diseases. Nevertheless, this product still bears a residual risk of infection because there are no absolutely safe test methods, test methods are not available for every disease, and the product might contain previously unknown pathogens. We therefore recommend considering all products containing human materials as potentially infectious. Exercise the same care in handling this product as you would in the handling of potentially infectious patient samples.

Storage life of the reconstituted calibrator: The reconstituted calibrator can be kept for up to 5 days if stored tightly sealed, light-protected and cool (+2 to +8 °C). For longer periods of storage (up to a maximum of 3 months), aliquot and deep-freeze below –18 °C.

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5.3 Reconstitution of the controls The urine controls for the normal range (order no. 0040) and the pathological range (order no. 0050) are subjected to the entire sample preparation, analogous to patient specimens. The prepared controls are included in every analytical series to monitor accuracy and precision within the system. To reconstitute the lyophilised urine controls, pipette exactly 8.0 ml distilled water into the vial. Let the vial stand at room temperature for about 10–15 min, shake occasionally and gently until the vial contents are completely dissolved. Avoid exposure to direct sunlight! The actual concentrations depend on the batch and will be found on the information leaflet accompanying the controls.

Caution: This product has been manufactured from pooled human urine. Each donor contributing to this product is constantly subjected to medical control and judged as free of infectious diseases. Nevertheless, this product still bears a residual risk of infection because there are no absolutely safe test methods, test methods are not available for every disease, and the product might contain previously unknown pathogens. We therefore recommend considering all products containing human materials as potentially infectious. Exercise the same care in handling this product as you would in the handling of potentially infectious patient samples.

Storage life of the reconstituted urine controls: The reconstituted urine controls can be kept for up to 5 days if stored tightly sealed, protected against light and cool (+2 to +8 °C). For longer periods of storage (up to a maximum of 3 months), aliquot and deep-freeze below –18 °C.

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5.4 Sample preparation procedure Stabilisation and predilution of urine samples: Transfer 3 ml of the urine to an appropriate vial and add 100 µl internal standard solution. Then dilute with 6 ml neutralisation buffer, and add 2 N NaOH until the colour changes from yellow to green or green-grey (see fig. 4); with very strongly acidified urines a more concentrated NaOH may be used. The green colour indicates that the pH value is correctly adjusted for the subsequent extraction. If the mixture shows a purple colour, the pH is too alkaline, and should be lowered by careful addition of 2 N HCl until the colour changes to green.

after addition of the neutralisation buffer

strongly acidic

pH correct

pH too alkaline

Fig. 4 pH-adjustment of the diluted sample Multiple acidic/alkaline pH-switches should be avoided, since this may lead to losses in recovery due to an increased salt loading. Sample extraction with Sample Clean Up Column: Briefly shake (resuspend) and label one Sample Clean Up Column for each sample. Remove the column cap and cut off the sealing nipple. Allow the equilibration buffer to flow out completely. Apply all of the diluted urine sample to the prepared Sample Clean Up Column. Discard the effluent. Washing: Rinse the Sample Clean Up Column with two column volumes of ultrapure water (HPLC grade): Fill the Sample Clean Up Column with ultrapure water (HPLC grade) to the upper rim of the funnel and let the water run through completely; repeat this step once. Discard the effluent. Elution: Place the Sample Clean Up Column on a correspondingly labelled test tube, add 6 ml elution buffer and collect the eluate. Acidify the eluate with 180 µl of 5 M HCl (30 µl 5 M HCl per ml eluate). Inject 20 µl of the eluate into the HPLC system.

too much NaOH + 2 N NaOH

+ 2 N HCl

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5.5 Sample preparation - overview Stabilisation and predilution of urine samples: 3 ml urine (patient sample, control, urine calibrator) + 100 µl Internal Standard solution + 6 ml Neutralisation Buffer Add 2 N NaOH until the colour changes from yellow to green or green-grey. If the urine mixture shows a purple colour (too alkaline) lower the pH value by careful addition of 2 N HCl until the colour changes to green. Extraction: Apply the entire volume of the diluted urine sample to the Sample Clean Up Column. Discard effluent. Washing: Fill up the Sample Clean Up Column to the funnel rim with ultrapure water (HPLC grade) and allow running through completely; repeat this step. Discard effluent. Elution: Add 6 ml Elution Buffer and collect eluate. HPLC analysis: Check flow rate, integration method and baseline. Acidify eluate with 180 µl of 5 M HCl (30 µl 5 M HCl per ml eluate). Inject 20 µl of the eluate. Please note: The Calibration Standard order no. 6003 is injected directly into the HPLC system, i.e. without preceding sample preparation!

5.6 Stability of the prepared samples (eluates) In the elution buffer used here catecholamines are stable for up to 48 h at +2 to +8 °C. For extended storage, the samples not acidified must be frozen below –18 °C.

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6 Data acquisition and evaluation

6.1 Calibration of the data analysis system Before beginning the quantitative analysis of the patient samples, it is recommended that a calibration chromatogram, containing all the substances of interest, should be run. For this purpose, inject the aqueous calibrator repeatedly, until two successive chromatograms show practically identical retention times, peak resolution and peak area/heights. These chromatograms can be used to set the integration parameters correctly. The chromatogram of the last test injection is used to calibrate the data analysis system (PC software, integrator). Enter the retention times obtained and the concentrations of the calibrator in the analysis table: a) Using the aqueous calibrator (order no. 6003):


ca. [min] Concentration

[µg/l]

Noradrenaline 4.5 25

Adrenaline 5.5 5

Internal standard (DHBA) 8.3 1

Dopamine 13.5 100

The aqueous calibrator is injected directly (without sample preparation) into the HPLC system. b) Using the urine calibrator (order no. 6009):


ca. [min] Concentration

[µg/l]

Noradrenaline 4.5 see information sheet

Adrenaline 5.5 - “ -

Internal standard (DHBA) 8.3 1

Dopamine 13.5 see information sheet

The urine calibrator is subjected to the entire sample preparation, analogous to patient specimens. The concentrations of the individual analytes depend on the batch and will be found on the information leaflet accompanying the standard. To ensure that no changes in either the calibration or the HPLC conditions (retention times etc.) have occurred, a further aliquot of the calibrator should be injected during and at the end of a series of analyses.

6.2 Quantitative evaluation with internal standard The use of an internal standard allows potential losses during sample preparation to be compensated for. A known amount of the internal standard is added to every specimen (calibrator, controls, patient specimens). The PC or integrator is given the appropriate peak (for catecholamine analysis peak no. 3 in the chromatogram of the calibrator) from the calibration run as the internal standard.

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Composition of the aqueous calibrator (order no. 6003): Noradrenaline: 25 ng/ml (25 µg/l) Adrenaline: 5 ng/ml (5 µg/l) Dopamine: 100 ng/ml (100 µg/l) Internal standard (DHBA): 50 ng/ml (50 µg/l) Concentration of the Internal Standard: 3,4 - Dihydroxybenzylamine (DHBA) 1.5 ng/µl 100 µl internal standard solution (1.5 ng/µl) are added to 3 ml urine. The final concentration of the internal standard in the sample then is 50 ng/ml (50 µg/l). Since this is identical with the calibrator, the concentration of the internal standard in all samples can be entered as “1“. When using the urine calibrator (order no. 6009) the same amount of internal standard is added to the calibrator as well as to the patient samples. Hence the concentration of the internal standard can be entered as "1" in all samples. The calibration and internal standard are traceable to reference substances purchased from a certified supplier. Calculation of creatinine-referred values (in µg/g creatinine): Divide the concentration value (in µg/l) by the concentration of creatinine in the urine (in g/l). Calculation of the 24 h excretion (in µg): Multiply the determined concentration (in µg/l) by the urine volume (in l).

7 Quality control Precision and accuracy of the analyses can be monitored by the inclusion of additional controls in each analytical run (Chromsystems urine controls, order nos. 0040 and 0050). If the analysis of these controls yields values outside the range given on the accompanying information leaflet, the system must be checked and, if necessary, recalibrated.

8 Reference ranges Noradrenaline: up to 570 nmol/24 h (up to 97 µg/24 h) Adrenaline: up to 150 nmol/24 h (up to 27 µg/24 h) Dopamine: up to 3240 nmol/24 h (up to 500 µg/24 h) Source [6]

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9 Conversion factors The following table lists conversion factors between mass and molar concentrations and conversely.

Analyte µg/l to nmol/l nmol/l to µg/l

Noradrenaline x 5.9102 x 0.1692

Adrenaline x 5.4585 x 0.1832

Dopamine x 6.5274 x 0.1532

10 Storage and lifetime of the reagents Unopened reagents can last up until the expiry date stipulated on the label, provided that the storage conditions which are indicated on the label are complied with. Storage conditions of the reagents:

Product Storage

Mobile Phase +18 bis +30 °C

Aqueous calibrator +2 to +8 °C

Urine calibrator below –18 °C

Internal Standard +2 to +8 °C

Neutralisation Buffer +18 bis +30 °C

Elution Buffer +18 bis +30 °C

Sample Clean Up Columns +18 bis +30 °C

Urine controls +2 to +8 °C

The reagents must be properly closed and stored directly after use. Provided that nothing else has been stipulated, the lifetime would then amount to one year after the date of opening, but will not exceed the expiry date. For Calibrator and Controls refer to chapter 5.2 and 5.3.

11 Waste disposal The mobile phase contains organic solvents. Dispose product residues into a container for organic halogen-free solvents. They must not be disposed together with domestic waste. Do not circulate into the main water supply. Dispose of in compliance with Directive 2008/98/EC on Waste and national and local requirements. The waste containers must be stored appropriately and only access permitted to authorized parties.

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12 Examples of chromatograms

12.1 Chromatogram of an aqueous calibrator

12.2 Chromatogram of a prepared urine sample

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13 Trouble shooting

Problem Possible causes Suggested remedies

Unstable baseline HPLC system contaminated with electrochemically active substances

Passivate pump, capillary and injection system with nitric acid

Cracks in the diaphragm Replace the Teflon adapter

Deposits on the diaphragm of the reference system

Clean the diaphragm with 25 % ammonia or replace it

Baseline fluctuates rhythmically

Damaged gaskets in the HPLC system pump units

Check pump for leaks, call service if necessary

Surface of the working electrode contaminated

Clean (activate) the working electrode

Damage to the interior wall of the detector cell

Replace the cell or have it serviced

Detector cell leaks Check cell for leaks: carefully tighten screw or replace gasket mask

Air bubbles in pump Check pump, remove air bubbles from the system

Baseline drifts System has not yet equilibrated Recycle mobile phase for a longer period

Environmental temperature changes

Provide constant environment temperature, thermostat column if frequent large temperature variations occur

Slight leak from one of the connections in the system

Tighten or replace capillary connector(s)

Background current high

System is contaminated with electrochemically active substances

Passivate pump, capillaries and injection system with nitric acid

Mobile phase is contaminated Replace with fresh mobile phase

HPLC column contaminated Replace column

Surface of working electrode is contaminated


Loss of detector sensitivity, signal loss

Surface of working electrode is contaminated


Working potential has fallen Check potential between working and reference electrode; if too low, service the reference electrode

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Problem Possible causes Suggested remedies

Interfering peaks in chromatogram

Injection system contaminated Rinse injection system with water, then with isopropanol; for cleaning manual injectors switch valves frequently during rinse phase. For rinsing an autosampler activate the injection process several times, follow the detailed cleaning information in the autosampler manual

Injection syringe contaminated Rinse syringe thoroughly with iso-propanol and water; rinse with 15–20 % nitric acid if necessary for a short time

System contaminated with electrochemically active substances

Passivate the pump, capillaries and injection system with nitric acid

Column contaminated Replace column

Inappropriate seal material injection system

Rotor seals in injection valves are usually made of Vespel which can leach with time, causing artefact peaks. Generally, rotor seals made of Tefzel should be used

Retention time changes Changes in room temperature Ensure a constant environmental temperature for column; thermostat column

Irregular flow rate Measure flow rate. If the pump flow is irregular, check the system for air bubbles; call service if necessary

Double peaks Dead volume in column inlet Replace column

Cracks in column bed Replace column

Dead volume in the injection system

Check the seating of the injection system loop; check the holes in the rotor seal

Peak shoulders Dead volume in the system Check capillary couplings for correct seating

Broad peaks, tailing Capacity of the column is exhausted

Replace column

High backpressure Particle accumulation in analytical column

Replace column or pre-column

Injection system blocked Rinse injection system with water, then with isopropanol and then again with water

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14 Literature 1. Cooper JR, Bloom RH. The biochemical basis of neuropharmacology. 5th edit, New York, Oxford

University Press (1986).

2. Wisser H, Knoll E (Greiling H, Gressner AM, Hsrg). Lehrbuch der Klinischen Chemie und Pathobiochemie. Schattauer Verlag Stuttgart (1987).

3. Bravo EL, Gifford RW Jr. (1984) Current concepts. Pheochromocytoma: diagnosis, localization and management. N Engl J Med 311(20): 1298-303.

4. Bravo EL. (1982) The clinical value of catecholamine measurement. Laboratory Management 20(6): 53-69.

5. Proye C, Fossati P, Fontaine P, Lefebvre J, Decoulx M, Wemeau JL, Dewailly D, Rwamasirabo E, Cecat P. (1986) Dopamine-secreting pheochromocytoma: An unrecognized entity? Classification of pheochromocytomas according to their type of secretion. Surgery 100(6): 1154-62.

6. Thomas L (Hrsg). Labor und Diagnose. 5. erweiterte Aufl, TH-Books Verlagsgesellschaft Frankfurt/Main (2000).

7. Ratge D, Baumgardt G, Knoll E, Wisser H. (1983) Plasma free and conjugated catecholamines in diagnosis and localisation of pheochromocytoma. Clin Chem Acta 132(3): 229-43.

8. Grobecker H, Saavedra JM, Dominiak P. Catecholamines in experimental and essential hypertension, in: The Heart in Hypertension, pp109-121. Springer Verlag Berlin, Heidelberg, New York (1981).

9. Folkow B. (1984) Plasma catecholamines as markers for sympathoadrenal activity in man. Introductory remarks. Acta Physiol Scand Suppl 527: 7-9.

10. Christensen NJ. (1979) Catecholamines and diabetes mellitus. Diabetologia 16(4): 211-24.

11. Lake CR, Sternberg DE, Van Kammen DP, Ballenger JC, Ziegler MG, Post RM, Kopin IJ, Bunney WE. (1980) Schizophrenia: elevated cerebrospinal fluid norepinephrine. Science 207(4428): 331-3.

12. Borg S, Kvande H, Sedvall G. (1981) Central norepinephrine metabolism during alcohol intoxication in addicts and healthy volunteers. Science 213(4512):1135-7.

13. Kauert G, Schoppek B, Clarmann v. M, Hibler A. (1989) Plasma-Katecholamin-Verlauf bei Alkylphosphat-Intoxikationen und deren Therapie. Klin Wochenschr 67: 456-62.

14. Darwish R, Elias AN, Vaziri ND, Pahl M, Powers D, Stokes JD. (1984) Plasma and urinary catecholamines and their metabolites in chronic renal failure. Arch Intern Med 144(1): 69-71.

15. Cohn JN, Levine TB, Olivari MT, Garberg V, Lura D, Francis GS, Simon AB, Rector T. (1984) Plasma norepinephrine as a guide to prognosis in patients with chronic congestive heart failure. N Engl J Med 311(13): 819-23.

16. Goldstein DS. (1984) Plasma catecholamines in clinical studies of cardiovascular diseases. Acta Physiol Scand Suppl 527: 39-41.

17. Elworthy PM, Hitchcock ER. (1986) Estimation of plasma catecholamines by HPLC with ECD in patients with subarachnoid haemorrhage. J Chromatogr 380(1): 33-41.

IM 6000 CATU EN 05/2016 R4

18. Ind PW, Causon RC, Brown MJ, Barnes PJ. (1985) Circulating catecholamines in acute asthma. Br Med J (Clin Res Ed) 290(6464): 267-9.

19. Halter JB, Stratton JR, Pfeifer MA. (1984) Plasma catecholamines in hemodynamic responses to stress states in man. Acta Physiol Scand Suppl 527: 31-8.

20. Hjemdahl P, Freyschuss U, Juhlin-Dahnfeld A, Linde B. (1984) Differentiated sympathetic activation during mental stress evoked by the stroop test. Acta Physiol Scan, Suppl 527: 25-9.

21. Weicker H. (1988) Determination of free and sulfoconjugated catecholamines in plasma and urine by HPLC. Int J Sports Med 9: 68-74 Suppl.

22. Pluto R, Bürger P. (1988) Normal values of catecholamines in blood plasma determined by HPLC with amperometric detection. Int J Sports Med 9: 75-78 Suppl.

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Appendix I:

Automated Sample Preparation with the Gilson® ASPEC™

ASPEC™- Racks The Chromsystems ASPEC™ files for catecholamines in urine require the following sample and reagent racks: ‒ Sample Rack, code 28 ‒ Solvent Rack, code 61 ‒ Mobile DEC Racks, code 101 (up to 3), with suitable Collect Racks and collection vials Setting Up the Racks

Reagent Rack Position

Samples Sample Rack 1–52

Empty vials Sample Rack 55–106

(same number as samples)

5 M HCl Sample Rack 107

Internal Standard (6004/A) Sample Rack 108

Neutralisation Buffer (6008/A) Solvent Rack 109

Elution Buffer (6006/A) Solvent Rack 110

Sample Clean Up Columns (6007/A) DEC Rack(s) 113–220

Collection vials Collect Rack(s) same number as SPE cartridges

Distilled water Reservoir

Before starting an analysis sequence the dilutor must be rinsed manually with solvent from the reservoir (Menu MANUAL - Prime dilutor)! Required Reagent Volumes: For each sample the following reagent volumes are required:

Reagent Volume

Internal Standard (6004/A) 55 µl

Neutralisation Buffer (6008/A) 2.75 ml

Elution Buffer (6006/A) 2.2 ml

5 M HCl 40 µl

Each solvent bottle must contain sufficient buffer volume for the number of samples to be prepared plus an additional volume of about 30 ml.

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Principle of the ASPEC™ Working Files The ASPEC™ mixes 660 µl of each urine specimen with 55 µl internal standard in a new empty vial, and adds Neutralisation Buffer for dilution and pH buffering. No manual pH adjustment is necessary prior to extraction! The diluted urine then is subjected to the complete sample preparation (SPE with wash and elution steps), and the obtained eluates are automatically acidified by the ASPEC™. The prepared samples are ready to inject into the HPLC system. The working file disk contains 2 files: "CATU_NI": ASPEC™ performs only the sample preparation; the samples are not injected into the HPLC system! "CATU": ASPEC™ must be properly connected to the HPLC system! This file controls the complete sample preparation for catecholamines in urine. The prepared eluates are automatically injected into the HPLC system, and the chromatography is started. During the HPLC run the next sample is prepared. The injection volume is set to 40 µl; it can be changed in step #15 - INJECT. The program step #18 - WAIT is used to coordinate the injection intervals according to the chromatography run time and the sample preparation time; it must be changed if necessary. Note: Samples with low catecholamine recovery (peak of the internal standard is significantly smaller than in the calibration standard) were prepared from too acidic urine. These specimens should be diluted with distilled water (or the pH value of the urine should be increased manually) and re-analysed.

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Configuration Set Up: These working files are designed for ASPEC™ control via its keypad. Some items in the CONFIG menu must be checked and, if necessary, adapted to the actual configuration of the ASPEC™ device. Configuration SAMPLER: Model Arm Rinsing station depth: e.g. 80 mm Rinsing station positions: A and/or B, C Injection loop(s): position and volume Calib. tubing volume ID number Configuration DILUTOR: Type Left syringe volume Right syringe volume Transfer tubing ID number

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Appendix II: Hazardous substance information The following information must be noted and the relevant safety measures taken. More information can be gathered from the respective material safety data sheets. These are available upon request or can be downloaded from our website www.chromsystems.com.

Pictograms Hazard and precautionary statements

Mobile Phase (order no. 5001/5002)

Warning

H302+H312+H332 Harmful if swallowed, in contact with skin or if inhaled.

H371 May cause damage to organs.

P280 Wear protective gloves/protective clothing/eye protection/face protection.

P301+P312 IF SWALLOWED: Call a POISON CENTER/doctor if you feel unwell.

P302+P352 IF ON SKIN: Wash with plenty of soap and water.

P403+P233 Store in a well-ventilated place. Keep container tightly closed.

Calibration Standard (order no. 6003)

Warning

H290 May be corrosive to metals.

Internal Standard (order no. 6004, 6004/A1, 6004/A5)

Warning

H290 May be corrosive to metals.

These components are not classified as dangerous according to European Union legislation:

Urine Calibration Standard (order no. 6009, 6009/T) Neutralisation Buffer (order no. 6055, 6008/A1, 6008/A5) Elution Buffer (order no. 6006, 6006/A9) Endocrine Urine Controls (order no. 0040, 0050) KCl solution, 3 mol/l (order no. 41239)

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Appendix III: Notes on manual calculation For manual calculation the following data are required: • Peak area/height of substance A in the chromatogram of the sample = ASample • Peak area/height of substance A in the chromatogram of the calibrator = ACalibrator • Peak area/height of the internal standard in the chromatogram of the sample = ISSample • Peak area/height of the internal standard in the chromatogram of the calibrator = ISCalibrator • The concentration of the substance A in the calibrator = CCalibrator The concentration of the substance A in the sample (CSample) is then calculated as follows:

CSample [µg/l] = ASample x ISCalibrator

x CCalibrator ACalibrator x ISSample

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Appendix IV: Validation To check the linearity and to validate the method, urine specimens were spiked with defined amounts of noradrenaline, adrenaline und dopamine. Multiple aliquots from these preparations were subjected to the sample preparation procedure. Recovery: The analytical recovery was determined from the slope of the calibration curves of spiked urine samples and diluted standard solutions:

Analyte Recovery

[%]

Noradrenaline 80

Adrenaline 68

Dopamine 91

Internal Standard 81

Linearity / limit of quantification: The method is linear from the designated limit of quantification up to the upper limit.

Analyte Limit of quantification

[µg/l] ca.* Linear range

up to at least [µg/l]

Noradrenaline 2.5 1450

Adrenaline 1.6 1800

Dopamine 3.6 2200

*The limits of quantification depend on the condition of the working electrode.

Intra-assay precision: Determination of the intra-assay precision was done by means of multiple clean up (n = 10) and determination of the analyte concentrations of the same specimen at 3 different concentrations:

Analyte Coefficient of variation [%] (at concentration in µg/l)

n = 10 n = 10 n = 10

Noradrenaline 1.2 (61.1) 1.7 (202) 1.2 (134)

Adrenaline 2.9 (11.7) 1.8 (47.9) 2.1 (30.5)

Dopamine 0.8 (193) 2.2 (453) 1.2 (326)

Inter-assay precision: Determination of the inter-assay precision was done by fourfold clean up and determination of the analyte concentrations in pooled urine (normal and pathological level) in 20 different test series in duplicate:

Analyte Coefficient of variation [%] (at concentration in µg/l)

n = 80 n = 80

Noradrenaline 2.5 (61.7) 3.1 (204)

Adrenaline 4.1 (11.9) 3.2 (49.8)

Dopamine 3.0 (192) 3.3 (450)

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Appendix V: Declaration of Conformity

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