TITLE : BOVINE URINE – DETECTION, DETERMINATION AND

CONFIRMATION OF CORTICOSTEROIDS – LC-MS/MS

1 OBJECTIVE AND SCOPE

Corticosteroids are administered to farm animals for the treatment of chronic inflammations and allergies. A side

effect of the use of corticosteroids is the suppression of the immune system.

On the Dutch market only dexamethasone is registered for the therapeutic use for bovine and equine animals.

Although corticosteroids are within the group B2f, within RIKILT they are treated as prohibited substances (group

A).

This SOP describes the detection (1 ion; no content, no confirmation), determination (1 ion; content, no

confirmation) or confirmation (2 ions) of corticosteroids in bovine urine. See also 7.3 for the performance.

The mentioned corticosteroids are detectable with this method from a level of at least 0.25 µg/l.

On the basis of RFS-0967-03, this method is also suitable for the determination and confirmation of prednisolone in

porcine urine from a level of 0.25 µg/l.

2 DEFINITION

SPE = Solid Phase Extraction

LC-MS/MS = liquid chromatography coupled with a tandem mass spectrometer

CE = Collision Energy

3 PRINCIPLE

With enzymatic hydrolyses conjugated corticosteroids present in the urine are converted into free compounds. After

hydrolyses the extract will be extracted with TBME. Thereafter a solid phase extraction on a polymer (Strata-X)

column takes place. The collected fraction will be evaporated, dissolved and analysed with a LC-MS/MS whereby

two specific transitions will be measured.

4 CHEMICALS AND REAGENTS

All chemicals should be of "pro-analysis" quality unless otherwise is stated. By water is meant,

Water purified through a Milli-Q system with a minimum resistance of 18.2 MΩ/cm or water of comparable quality.

Reference to a product and/or manufacturer is only for information and identification and does not exclude any

other products and/or manufacturers which also may suit.

4.1 Standards

4.1.1 Beclomethasone (Sigma B0385)

4.1.2 Betamethasone (Sigma B7005)

4.1.3 Clobetasol (Steraloids P0086-00)

4.1.4 Dexamethasone (Sigma D1756)

4.1.5 Flumethasone (Sigma F9507)

4.1.6 Isoflupredone (Delta fludrocortisone; Steraloids P0553-000)

4.1.7 Methylprednisolone (Dr. Ehrenstorfer 15141700)

4.1.8 Prednisolone (Sigma P6004)

4.1.9 Prednisone (Dr. Ehrenstorfer 16286550)

4.1.10 Triamcinolone acetonide (Dr. Ehrenstorfer 17635000)

4.1.11 Cortisol (Hydrocortisone; Sigma H4001)

4.1.12 Cortisone (Sigma C-2755)

4.1.13 Dexamethasone-d4 (CDN D-5559)

4.1.14 Triamcinolone acetonide-d6 (RIVM, self-synthesized)

4.1.15 Prednisolone-d8 (TRC P 703742)

4.2 Chemicals

4.2.1 Ammonia 25% (Merck 1.05432)

4.2.2 Sodium hydroxide (Merck 1.06498)

4.2.3 Acetic acid (Merck 1.00063)

4.2.4 β-Glucuronidase/arylsulfatase (Merck 4114)

4.2.5 Methanol (Biosolve 13683502)

4.2.6 Acetonitrile (Biosolve 01203502)

4.2.7 Tert-Butyl methyl ether (Merck 1.01849)

4.2.8 Sodium acetate anhydrous (Merck 6268)

4.3 Standard solutions

4.3.1 Main standard solution corticosteroids (1000 mg/l)

Make main standard solutions of the individual corticosteroids (4.1.1 up to and including 4.1.12) by weighing 3 – 10

mg of each corticosteroid on an analytical balance with an accuracy of 0.01 mg. Calculate the amount of methanol

needed for a concentration of 1000 mg/l, taking into account the purity and appearance of the standard. Add the

calculated amount of methanol to the standard. Keep the solutions in the freezer and in the dark. Under these

conditions the shelf life of cortisone, betamethasone and beclomethasone is 1 year and for prednisone,

prednisolone, cortisol, isoflupredone, methylprednisolone, dexamethasone, flumethasone, triamcinolone acetonide

and clobetasol 3 years.

4.3.2 Main standard solution internal standards (1000 mg/l)

Make main standard solutions of the individual internal standards (4.1.13 and 4.1.15) by weighing 3 – 10 mg of

each standard on an analytical balance with an accuracy of 0.01 mg. Calculate the amount of methanol needed for

a concentration of 1000 mg/l, taking into account the purity and appearance of the internal standard. Add the

calculated amount of methanol to the standard. Keep the solutions in the freezer. Under these conditions the shelf

life of these solutions is 1 year.

4.3.3 MSO I (10.0 mg/l)

Pipette 100 µl of each of the main standard solutions corticosteroids (4.3.1) into a graduated flask of 10 ml. Fill the

flask with methanol up to 10 ml and mix. Keep the solutions in the freezer. Under these conditions the shelf life of

these solutions is 1 year.

4.3.4 MSO II (0.1 mg/l)

Pipette 100 µl of the MSO I (10.0 mg/l) (4.3.3) into a graduated flask of 10 ml. Fill the flask with methanol up to 10

ml and mix. Keep the solution in the freezer. Under these conditions the shelf life of this solution is 3 months.

4.3.5 MSO III 0.025 mg/l)

Pipette 2,5 ml of the MSO II (0.1 mg/l) (4.3.4) into a graduated flask of 10 ml. Fill the flask with methanol up to 10

ml and mix. Keep the solution in the freezer. Under these conditions the shelf life of this solution is 3 months.

4.3.6 MSO IV cortisone/cortisol (1.0 mg/l)

Pipette 25 µl of the main standard solutions cortisol and cortisone (4.3.1) into a graduated flask of 25 ml. Fill the

flask with methanol up to 25 ml and mix. Keep the solution in the freezer. Under these conditions the shelf life of

this solution is 3 months.

4.3.7 ISO I (10 mg/l)

Pipette 100 µl of each of the main standard solutions internal standards (4.3.2) into a graduated flask of 10 ml. Fill

the flask with methanol up to 10 ml and mix. Keep the solutions in the freezer. Under these conditions the shelf life

of these solutions is 1 year.

4.3.8 ISO II (0.1 mg/l)

Pipette 100 µl of the ISO I (4.3.7) into a graduated flask of 10 ml. Fill the flask with methanol up to 10 ml and mix.

Keep the solution in the freezer. Under these conditions the shelf life of this solution is 3 months.

4.3.9 Working standard initial test 5 µg/l (which corresponds to the end extract MMS (0.25 µg/l))

See 4.3.10 standard 5 µg/l.

4.3.10 External calibration line corticosteroids (0.0 – 1000 µg/l) (which corresponds to the end extract MMS 0 –

MMS 50 µg/l)

Because in most urines prednisolone, cortisone and cortisol occurs naturally, also an external calibration line is

measured with the analysis of the sample series. The concentrations of the calibration line are based on the

concentrations which occur naturally in urine samples (the contents of cortisone and cortisol can be well over 100

µg/l). Only for cortisol and cortisone additional calibration points from 5 till 50 µg/l (based on the MMS end extract)

are measured.

Pipette for each calibration point an amount of (internal) standard solution in a vial of 2 ml according Table 2.

Evaporate till it is dry and dissolve in water/acetonitrile 75:25 v/v according Table 2.

For each measurement series a fresh calibration line is made.

Table 2. Addition scheme

Standard

(µg/l)

Standard

relative to end extract MMS

(µg/L)

MSO II (0.1 mg/l) (4.3.4)

(µl)

MSO IV Cortisol/cortisone (1.0 mg/l) (4.3.6)

(µl)

ISO II (0.1 mg/l) (4.3.8)

(µl)

After evaporation dissolve in (µl)

Water/acetonitrile 75:25

0,0 0,0 0 - 40 200

2,5 0,125 10 - 80 400

5,0 0,25 10 - 40 200

10,0 0,5 20 - 40 200

20,0 1,0 40 - 40 200

40,0 2,0 80 - 40 200

100 5,0 - 20 40 200

200 10,0 - 40 40 200

500 25,0 - 100 40 200

1000 50,0 - 200 40 200

4.4 Reagents

4.4.1 Acetate buffer 2 M, pH 5.2

Weigh 129.5 grams ± 0.5 gram of sodium acetate (= 1.58 mole) in a beaker of 1 litre and ad about approx. 900 ml

of water. Adjust the pH to 5.2 ± 0.05 after dissolution with acetic acid, for this approx. 25 ml (= 0.42 mole) is

needed. Add water to the mark and mix. The shelf life of this solution is 3 months at room temperature.

4.4.2 Acetate buffer 0.2 M, pH 5.2

Mix 100 ml of acetate buffer 2 M pH 5.2 (4.4.1) with 900 ml of water. Check the pH and if necessary adjust with

acetic acid to pH 5.2 ± 0.05. The shelf life of this solution is 3 months at room temperature.

4.4.3 Water / acetonitrile / 0.1% acetic acid 75:25 (v/v)

Mix 25 ml of acetonitrile with 75 ml of water and add 0.1 ml of acetic acid. The shelf life of this solution is 3 months

at room temperature.

4.4.4 Mobile phase A: 0.1% acetic acid in water

Mix 1 litre of water with 1 ml of acetic acid. The shelf life of this solution is 3 months at room temperature.

4.4.5 Mobile phase B: 0.1% acetic acid in acetonitrile

Mix 1 litre of acetonitrile with 1 ml of acetic acid. The shelf life of this solution is 3 months at room temperature.

4.4.6 2% Ammonia in water

Mix 80 ml of ammonia 25% with 920 ml of water. The shelf life of this solution is 3 months at room temperature.

4.4.7 Methanol / ammonia 2% 45:55 (v/v)

Mix 450 ml of methanol with 550 ml of 2% ammonia. The shelf life of this solution is 3 months at room temperature.

5 EQUIPMENT

References to a product and / or manufacturer are for information only and do not exclude other products and / or manufacturers which may also suit. 5.1 Analytical balance

With a minimum weighing range of 0 to 10 g, with an accuracy of 0.01 mg (Mettler AT 261)

5.2 Top loading balance

With a minimum weighing range of 0 to 1500 g, with an accuracy of 0.01 gram (Mettler PB 1502)

5.3 Water bath (Julabo SW-20C) 5.4 Centrifuge (Eppendorf 5810)

5.5 Evaporator (Turbo-Vap LV evaporator Zymark)

5.6 Vortex (IKA minishaker MS2)

5.7 Ultrasonic table top cleaner (Sonicor)

5.8 Several pipettes suitable for aqueous and/or organic solutions (e.g. Gilson pipettes or Eppendorf Multipette)

5.9 pH-indicator strips pH = 4 – 7 (Merck 9542)

5.10 Round bottom tubes of 10 ml (RB 100 x 15/16 mm, IMZ 31.00.56)

5.11 Polypropylene tubes of 12 ml with screw cap (Greiner 163275)

5.12 LC vial without insert with screw cap and pre-slit septum (Grace 8625313)

5.13 LC vial with 300 µl insert with screw cap and pre-slit septum (Grace 8625343)

5.14 Vacuum manifold for SPE columns (Alltech 210224)

5.15 Vacuum pump (KNF Lab laboport UN 842.3FTP)

5.16 SPE Strata-X columns (60 mg/ 3 ml, Phenomenex 8B-S100-UBJ)

5.17 Parafilm (American National Can)

5.18 Pasteur pipettes of 3 ml (Copan)

5.19 LC-MS system consisting of the following or similar components

5.19.1 Waters Acquity Ultra Performance LC delivery and injection system

5.19.2 Analytical column: Acquity UPLC BEH C18, 1.7 µm, 100 x 3 mm (Waters part.no. 186002352)

5.19.3 Mass spectrometer: Micromass Quattro UltimaTM Pt provided with an ESI interface

6 PROCEDURE

6.1 General

This SOP describes the determination of corticosteroids in bovine urine. The data will be filled in the observation

form. An example of the observation form is shown in Appendix 2. A checklist of the method is shown in Appendix

3. An example of a chromatograph is shown in Appendix 4.

6.2 Safety precautions

It is important to work as much as possible in a safety cabinet. In this way, inhalation of and skin contact with

standards and solvents will be avoided as much as possible. Use a lab coat and if necessary, use gloves (F0071).

6.3 Pre treatment

Frozen urine samples are thawed at room temperature. A sample is homogenized before an aliquot is taken. The

original samples are stored in a freezer.

6.4 Amount of sample

The amount of sample which is taken into preparation is 2 ml.

6.5 Description procedure

6.5.1 Composition of a sample series

A series consist of 6 matrix matched standards (MMS) and a number of samples. The MMS are based on a urine

sample in which in previous analysis no non-natural corticosteroids are determined.

6.5.2 Preparing matrix matched standards (MMS)

Pipette 6 portions of 2.0 ml of blank urine in 6 separate tubes of 12 ml.

Add standard solutions and internal standard solutions to the blank urine according Table 3.

Table 3. Preparing MMS

MMS

MSO III (0.025 mg/l) (4.3.5) (µl)

MSO II (0.1 mg/l) (4.3.4) (µl)

ISO II (0.1 mg/l) (4.3.8) (µl)

MMS 0.0 µg/l 0 - 20 MMS 0.125 µg/l 10 - 20 MMS 0.25 µg/l 20 - 20 MMS 0.5 µg/l - 10 20 MMS 1.0 µg/l - 20 20 MMS 2.0 µg/l - 40 20

6.5.3 Preparing 1st line control

Use a urine in which was found to be negative in previous analysis. Pipette 2.0 ml of this blank sample in a tube of

12 ml. Add an amount of standard solution and internal standard solution as for MMS 0.25 µg/l.

6.5.4 Preparing samples

Pipette 2.0 ml of each sample in separate tubes of 12 ml. Add 20 µl of ISO II (0.1 mg/l, 4.3.8) to all samples.

6.5.5 Hydrolyses

Add 1 ml of acetate buffer pH 5.2 to all samples (4.4.1) and mix. Check the pH with pH indicator strips. If necessary

adjust the pH to 5.2 ± 0.2 with a few drops of (diluted) acetic acid.

Add 25 µl of β-glucuronidase / arylsulfatase (4.2.4) and mix. Close the tubes and place them for 2 hours in a water

bath at 55°C.

6.5.6 LLE extraction

Cool down the samples till room temperature after hydrolyses.

Add 6 ml of TBME and shake the tubes during 30 seconds.

Centrifuge for 2 minutes at 2500 g and transfer the TBME supernatant into a clean tube.

Repeat the TBME extraction and add the TBME layer to the TBME of the first extraction.

Evaporate in an evaporator at 55°C ± 5°C till it is dry.

Dissolve the residue in 1 ml of acetonitrile and sonicate during 2 minutes.

Add 3 ml of 0.2 M acetate buffer pH 5.2 (4.4.2) and mix.

6.5.7 SPE purification

- Condition one SPE column for each sample in succession with 3 ml of methanol and 3 ml of water.

- Transfer the sample extract to the column and let it go through the column drop by drop.

- Wash the column 2 times with 3 ml of methanol / 2% ammonia 45:55 (v/v).

- Wash the column with 3 ml of water and dry the columns for approx. 2 minutes under vacuum.

- Eluate with 3 ml of methanol and collect in a glass tube.

- Evaporate with an evaporator at 55°C ± 5°C till it is dry.

- Dissolve the residue in 100 µl water / acetonitrile / 0.1% 75:25 (v/v), mix and sonicate during 2 minutes.

- Centrifuge the samples for 5 minutes at 2500 g.

- Transfer the end extract into a LC vial with insert.

6.5.8 LC-MS/MS analysis

6.5.8.1 Measurement conditions LC

Analytical column: Acquity UPLV BEH C18, 1.7 µm, 100 mm x 2.1 mm

Mobile phase A: 0.1% acetic acid in water

Mobile phase B: 0.1% acetic acid in acetonitrile

Flow: 0.4 ml/min

Injection volume: 20 µl

Sample tray: 12°C

Column temperature: 40°C

Run time: 9 min

Solvent delay: 0 till 1.5 min and 5.3 till 9 min

Table 4. Gradient LC

Time (min)

Mobile phase A (%) Mobile phase B (%)

0 75 25 0.5 75 25 0.7 70 30 3.0 70 30 5.5 0 100 7.0 0 100 7.1 75 25 9.0 75 25

6.5.8.2 MS conditions

Ionisation mode: ESI, positive

Capillary voltage: 3.2 kV

Cone voltage: 40 V

Source temperature: 120°C

Desolvation temperature: 350°C

Desolvation gas flow: 780 l/hour

Cone gas flow: 120 l/hour

CID gas: Argon (purity ≥99.998), p = 3 x 10-3 mbar

The corticosteroids fragment into structure related fragments. The theoretical mono isotopic masses of the

precursor ions and the associated product ions are shown in Table 5. The allowed deviations of the mass settings

are ± 0.2 amu.

The guideline values of the fragmentation conditions per component are also shown.

Table 5. MS/MS Fragmentation conditions

Component Precursor ion mass (m/z)

Product ion mass (m/z)*

Collision energy (eV)

Window (min)**

Prednisone 359.3 237.3 323.3

16 8

1.5 – 2.8

Prednisolone-d8 369.3 351.2 8 1.5 – 2.8

Prednisolone 361.3 343.3 325.2

8 8

1.5 – 2.8

Cortisol 363.3 121.2 327.2

20 13

1.5 – 2.8

Isoflupredone 379.3 359.2 341.2

8 10

1.5 – 2.8

Cortisone 361.3 163.1 121.1

23 28

1.5 – 2.8

Methylprednisolone 375.2 339.2 161.1

8 18

2.8 – 3.9

Betamethasone 393.3 373.2 355.2

8 10

2.8 – 3.9

Dexamethasone-d4 397.3 377.3 8 2.8 – 3.9 Dexamethasone 393.3 373.2 8 2.8 – 3.9

355.2 10

Flumethasone 411.3 253.2 391.3

13 8

2.8 – 3.9

Beclomethasone 409.2 391.2 373.2

10 8

3.7 – 4.5

Triamcinolone acetonide-d6 442.3 422.3 8 3.7 – 4.5

Triamcinolone acetonide 435.3 415.4 339.3

8 13

3.7 – 4.5

Clobetasol 411.3 391.3 355.3

5 10

4.5 – 5.3

* The product ion with the highest intensity is underlined

** This window can be dependent of the system and column. The given values are therefore indicative.

6.5.8.3 Initial test LC-MS/MS system

A test analysis will be performed prior to the start of the analytical series in order to confirm the proper operation of

the LC-MS/MS system and the suitability of the used standards. First inject the working standard (4.3.9) at the LC-

MS/MS system. With this standard the retention time of the components is determined at the system and the time

window (Table 5) is set correctly. Check if the signal/noise ratio of the ion with the least intensity of each

corticosteroid is equal or more than the value in the observation form. Register this on the observation form

conform Appendix 2. When this criterion is met, the analytical series can be started. When an abnormality is found,

consultation with the responsible supervisor will follow about the procedure to be followed.

6.5.8.4 Sequence analysis sample series

Recommended injection sequence after the initial test:

- External calibration line (4.3.10)

- Blank solvent

- MMS series (6.5.2)

- Blank solvent

- 1st line control (6.5.3)

- Samples (6.5.4)

- Blank solvent

- External calibration line (4.3.10)

- Blank solvent

- MMS series (6.5.2)

- Blank solvent

6.6 Data storage and processing

The LC-MS data will be stored in a designated directory at the server with a name which corresponds with the date

of analysis [4]. Processing and interpretation of the data will be done with Masslynx software. The data of the initial

test and the analysed samples will be shown on the observation form conform Appendix 2 of this SOP. The

electronic versions of the actual observation forms can be found on the network of the cluster ‘DBM’.

7 RESULTS

7.1 Calculations

This chapter describes which comparisons are used for the various parameters in the observation form. Also the

criteria for the assessment of the analysis are described. The observation form used for the calculation of the

parameters can be found on \Waarnemingsformulieren\SOPA1137.xls .

The components prednisone, prednisolone, cortisone, cortisol, isoflupredone and methylprednisolone will be

calculated using the internal standard prednisolone-d8.

The components betamethasone, dexamethasone and flumethasone will be calculated using the internal standard

dexamethasone-d4.

The components beclomethasone, triamcinolone acetonide and clobetasol will be calculated using the internal

standard triamcinolone acetonide-d6.

For quantification of prednisolone, cortisol and cortisone an external calibration line will be used, for all the other

components the MMS calibration line will be used.

The following definitions and comparisons are used in this chapter.

Comparison I: Calculation of the ion ratio (R)

%100×

=

high

low

A

AR

Wherein:

R = ion ratio (%)

Alow = peak area of the product ion with the lowest intensity

Ahigh = peak area of the product ion with the highest intensity

Comparison II: calculation of the relative deviation of the ion ratio (D)

( )%100

. ×

−=

mean

meansample

R

RRD

Wherein:

D = relative deviation of the ion ratio of the sample relative to the average ion ratio of the MMS series 0.25

µg/l up to 2.0 µg/l (50.0 µg/l) prior and after the series (%)

Rsample = ion ratio of a component in the sample (%)

Rmean = mean ion ratio of the MMS series 0.25 µg/l up to 2.0 µg/l (50.0 µg/l) prior and after the series (%)

Comparison III: Calculation of the response factor (RF)

=

IS

compound

Area

AreaRF

Wherein:

RF = response factor

Areacomponent = sum of the peak area of the product ions

AreaIS = peak area of the product ion van de internal standard *

Comparison IV: Calculation relative retention time (RRT)

=

IS

analyte

RT

RTRRT

Wherein:

RRT = relative retention time of the component relative to the internal standard (%)*

RTanalyte = retention time of the component (minutes)

RTIS = retention time of the internal standard (minutes)*

Comparison V: Calculation of the relative deviation of the relative retention time (∆RRT)

%100×

−=∆

mean

meansample

RRT

RRTRRTRRT

Wherein:

∆RRT = relative deviation of the relative retention time of a component relative to the average relative

retention time of the MMS series 0.25 µg/l tot 2.0 µg/l (50.0 µg/l) prior to and after the series (%)

RRTsample = relative retention time of a component in the sample

RRTmean = mean relative retention time of a component in the MMS series 0.25 µg/l tot 2.0 µg/l (50.0 µg/l)

prior to and after the series (%)

Comparison VI: Calculation content of the sample (X)

Wherein: X = content of a component in the sample (µg/l)

RF = response factor (comparison III)

b = intercept of the calibration line with the y–axis (resulting from linear regression*)

a = slope of the calibration line (resulting from linear regression*)

* Plot the response factors prior to and after the samples as a function of the added content. Perform linear regression on it

according the least squares method.

Comparison VII: Calculation minimal response of the internal standards to ensure the detection limit 0.25 µg/l

Wherein:

Amin = minimal surface area of the internal standard in a sample whereby the detection level at a level

of 0.25 µg/l is ensured

A mms = lowest surface area of the internal standard of MMS 0.25 µg/l prior to and after the samples

S/N = lowest signal/noise ratio of the product ion of MMS 0.25 µg/l prior to and after the samples

7.2 Criteria

For acceptance of the run the parameters specified in section 7.1 are checked with the limits specified in this

section. The limit values are referred to as criteria. All these parameters are automatically determined when the

observation form is filled in (see Appendix 2). Herein it is also indicated automatically if the parameters meet the

criteria. When a calculated parameter does not meet the criteria as described in this section, appropriate measures

are taken in consultation with the responsible supervisor.

7.2.1 Drift response factor

−=a

bRFX

NS

AA mms

/

*6

min=

The comparison of the MMS or external calibration line prior to and after the samples is indicative for the proper

function of the system during the entire analytical run. Plot for both series MMS or external calibration line the

response factor as a function of the added content. Perform linear regression on the individual data sets according

to the least squares method. Two slopes will follow out of this. The slopes of both series MMS or external

calibration lines may not differ by more than 20%.

7.2.2 Linearity

The MMS or external calibration line is used to determine the linearity of the LC-MS/MS system and to verify that

the procedure of the samples was correct. Calculate the correlation coefficient of the calibration lines (7.2.1). The

correlation coefficient of both series has to be greater than or equal to 0.99.

7.2.3 Accuracy 1st line control

For dexamethasone, flumethasone, beclomethasone and triamcinolone acetonide control charts are used.

Quantification is based on the peak area of both product ions. Calculate the response factor of each corticosteroid

in the 1st line sample conform comparison III (7.1). Calculate the content of corticosteroids in the 1st line control with

comparison VI (7.1) (a and b are known from linear regression of the MMS or external calibration line prior to and

after the samples). Calculate the accuracy by expressing the calculated content as a percentage of the real added

content.

Fill in the calculated accuracy in the control chart. For the accuracy, the criteria described in SOP F0057 [3] for

control charts are applied.

7.2.4 Sensitivity

In each series of analysis is verified whether the sensitivity is achieved with MMS 0.25 µg/l. Check whether the

signal/noise ratio of the ion of the lowest intensity of each corticosteroid is greater than or equal to the value

described in the observation form. Register this on the observations form conform Appendix 2.

7.2.5 Maximum deviation ion ratio

Determine the average ion ratio of the corticosteroids in the MMS series 0.25 µg/l up to 2.0 µg/l (comparison I).

Calculate the deviation of the ion ratio of the individual MMS samples relative to the calculated average ion ratio

(comparison II). The calculated deviation of the individual MMS samples has to meet the EU criteria [1] as listed in

Table 6.

Table 6: Maximum allowed relative deviation of the ion ratio according to EU-criteria [1]

Ion ratio (R) Allowed relative deviation (D)

R > 50% ≤ 20%

20% < R ≤ 50% ≤ 25%

10% < R ≤ 20% ≤ 30%

R ≤ 10% ≤ 50%

7.2.6 Maximum deviation relative retention time

Determine the average relative retention time for the MMS 0.125 µg/l up to and including 2.0 µg/l. The deviation in

the individual MMS samples compared to the average relative retention time should not be more than 2.5%.

7.2.7 Guarantee confirmation limit in a sample

On the basis of the signal of the internal standard for each sample is determined whether the detection at the level

of the reporting limit (0.25 µg/l) is ensured. Calculate the minimum surface area in a sample whereby quantification

at the target value is ensured. Hereby comparison VII is used.

The surface area of the internal standard in a sample should be more than or equal to the calculated value. If the

criterion is not met, the sample should be re-analysed, unless a signal from the lowest product ion is more than or

equal to S/N=3 is detected in the sample.

7.2.8 Quantification

Quantification is based on the peak area of both product ions. Calculate the response factor of each corticosteroid

in a sample conform comparison III (7.1). Calculate the content of the corticosteroids in each sample with

comparison VI (7.1).

The content of each corticosteroid in the sample is expressed in µg/l. The content is calculated automatically in the

concerning observation forms (example in Appendix 2).

7.2.9 Criteria for the assessment of the individual samples

Calculate for all the individual samples in which a signal for a corticosteroid is found the ion ratio, the relative

deviation of the (relative) retention time and response factor.

The identity of a corticosteroid in a sample is confirmed when the relative deviation of the ion ratio meet the criteria

listed in Table 6 and the relative retention time does not deviate by more than 2.5% of the average relative

retention time in MMS 0.125 µg/ up to and including 2.0 µg/l prior to and after the samples.

7.3 Final results

Table 7: Reporting in LIMS: (in Dutch)

No signal

And/or

not confirmed

Content ≥ CCα

and

confirmed

Determined

no CCα

confirmed

Prednisone na * a

Prednisolone na * a

Prednisolone (porcine

urine)

<0,125 Calculated content n.v.t.

Isoflupredone na * a

Methylprednisolone na * a

Betamethasone na * n.v.t.

Dexamethasone < 0,125 Calculated content n.v.t.

Flumethasone < 0,125 Calculated content n.v.t.

Beclomethasone < 0,125 Calculated content n.v.t.

Triamcinoloneacetonide < 0,125 Calculated content n.v.t.

Clobetasol na * a

Cortisol na * a

Cortisone na * a

*No CCα for the concerning standards

When the deviation of the relative retention time and the deviation of the ion ratio meet the criterion and the content

of a corticosteroid in a sample ≥ CCα, the calculated content will be reported.

When a corticosteroid is not determined or the calculated content of a corticosteroid in the sample is < CCα, the

concerning content will be reported as < CCα.

8 REGISTRATION

All relevant data, of the procedure, is listed in the lab journal, with reference to the raw data folder (number). All raw

data are stored digital in the mentioned raw data folder.

LITERATURE

[1] Final Draft Version of Revision of EC Directive 93/23/EC, SANCO/1085/2000, revision 7, 2002

[2] SOP F0071 – Personal protective equipment within RIKILT

[3] SOP F0057 ‘Controlekaarten - Gebruik en beheer’

[4] SOP T0359 – ‘Bediening en onderhoud – Micromass Quatro Ultra Platinum’

[5] EC Directive 2002/657/EC

APPENDIX 1 FOR SOP-A-1137 Performance Sheet bovine urine

Validation conform F0052 (A0400) Analyt

Concentr

ation

inte

rval / ra

nge

Unit

Accura

cy /

Tru

eness (

%)

Rel.st.a

fw.

jaccura

cy (

%)

Rel. s

td. a

fw.

Repeata

bili

ty (

%)

CCα

CC

β

Beclomethasone 0.25 µg/l 90** 18,4** 11,2

0.50 µg/l 86 10,9 10,9 0,58 0,65

0.75 µg/ 87 12,7 12,7 Betamethasone ** ** * *

Clobetasol * *

Dexamethasone 0.25 µg/l 95** 15,9** 12,2 0.50 µg/l 95 10,9 8,8 2,11 2,23

0.75 µg/l 96 9,8 6,9

Flumethasone 0.25 µg/l 95** 16,6** 16,3 0.50 µg/l 98 15,1 14,9 0,62 0,74

0.75 µg/ 101 15,6 15,0

Isoflupredone * * Methylprednisolone * *

Prednisolone * *

Prednisone * * Triamcinolone acetonide 0,25 µg/l 102** 13,7** 5,0

0,5 µg/l 98 8,7 8,6 0,57 0,64

0,75 µg/l 96 5,7 5,6

Cortisol * * Cortisone * *

* Due to a high RSD the quantification is not reliable, zo no validation data are shown.

** Adjusted after evaluation of the 1st line control in July 2017.

LINEARITY, SPECIFICITY, RUGGEDNESS AND STABILITY: See validation dossier OTHER INFORMATION: Information concerning the validation can be found in:

- Lab journal 2412 page 11, 19, 21, 24, 28, 31 and 32 - Raw data file GB 11-046

For dexamethasone is a recommended concentration (RC) of 2.0 µg/l. De CCα and CCß are calculated as follows:

CCα = 2 + 1.64*srl CCß = CCα + 1.64* srl (srl op 0.75 µg/l level)

For the other corticosteroids no RC or MR(P)L is given. For these substances a target value of 0.5 µg/l is used for validation.

The CCα and CCß are calculated as follows:

CCα = 0.5 + 1.64* srl CCß = CCα + 1.64* srl (srl op 0.5 µg/l level)

Pe

rform

an

ce

Sh

ee

t po

rcin

e u

rine

V

alid

atio

n c

onfo

rm F

005

2 (A

040

0)

Analy

t

Concentration interval / range

Unit

Accuracy / Trueness * (%)

Rel.st.dev. accuracy (RSDRL-%)

Recovery (%)

RSDr (%)

Systematic deviation (%) (%)

St. dev. Repeatability (sr)

Repeatability (r)

St. dev. within-lab reproducibility (RL)

Within-lab Reproducibility

Expanded measurement uncertainty

Reproducibility

LOD

LOQ

CCα**

CCβ

Pre

dnis

olo

ne

2

µg/l

119

13.7

8.6

0.2

0

0.5

7

0.3

3

0.9

1

0.5

4*

2.5

3.1

* =

1.6

4 x

St. d

ev. W

ithin

-lab re

pro

ducib

ility.

** CCα

and C

Cß

are

calc

ula

ted a

bove th

e ta

rget v

alu

e o

f 2 µ

g/l.

APPENDIX 2 for SOP-A-1137 OBSERVATION FORM BIJLAGE bij SOP A1137

A 1137-v1

Analysedatum:

Matrix: Urine

Component: Cortisol rapportagegrens (µg/l) = 0.05

Initiële test

Concentratie Ionratio

Data file Omschrijving RT Area m/z 363 > 121 Area m/z 363 > 327 (µg/L)

0.25

Criteria Initiële test

Omschrijving S/N m/z 327 Voldoet Serie gestart: Paraaf:

Gevoeligheid m/z S/N ≥ 20 Datum:

Standaard.

Standaard geanalyseerd vóór monsters

Data file Omschrijving RT Area m/z 363 > 121 Area m/z 363 > 327 RT IS Area IS Concentratie Relatieve Respons Afwijking Ionratio Afwijking Gehalte Waarde

Pred-D8 m/z 351 (µg/l) RT factor rel. RT (%) ionratio (%) (µg/kg) (µg/l)

0.0 Richting

0.125 Snijpunt y-as

0.25 Correlatie

0.5

1.0

2.0

Standaard geanalyseerd ná monsters Gemiddelde area IS vóór:

Data file Omschrijving RT Area m/z 363 > 121 Area m/z 363 > 327 RT IS Area IS Concentratie Relatieve Respons Afwijking Ionratio Afwijking Gehalte Waarde

Pred-D8 m/z 351 (µg/l) RT factor rel. RT (%) ionratio (%) (µg/kg) (µg/l)

0.0 Richting

0.125 Snijpunt y-as

0.25 Correlatie

0.5

1.0

2.0

Waarde

Gemiddelde area IS ná:

Verloop gevoeligheid serie Gem rel. RT Gem ionratio Richting

Verloop responsfactor Stdev rel. RT Stdev ionratio Snijpunt y-asGemiddelde area IS RSD rel. RT RSD ionratio Correlatie

MMRS

RT IS Area IS Concentratie Relatieve Respons Afwijking Ionratio Afwijking Terugvinding

Data file Omschrijving RT Area m/z 363 > 121 Area m/z 363 > 327 Pred-D8 m/z 351 (µg/l) RT factor rel. RT (%) ionratio (%) (%)

Criteria voor runacceptatie:

Waarde Voldoet Serie geaccepteerd: 1e validatieniveau

≤ 20.0% Datum: Paraaf:

≥ 0.990

N.v.t. N.v.t.

S/N ≥ 6

=< 2e validatieniveau

≤ 2.5% Paraaf:

Criteria voor acceptatie monster resultaat:

≤ 2.5%

≤

Monsters

Data file Omschrijving RT Area m/z 363 > 121 Area m/z 363 > 327 RT IS Area IS Borging det. S/N Relatieve Afwijking Respons Ionratio Afwijking Gehalte Confirmatie

Pred-D8 m/z 351 1/4*MRL Area m/z laagste intensiteit RT rel. RT (%) factor ionratio (%) (µg/l) resultaat

Opmerkingen

Max. afw. ionratio conform EU-criteria

Max. afw. RT conform EU-criteria

Min. signaal IS voor borging detectie op rapportagegrens

Gevoeligheid m/z 325 Std 0.25 µg/l

CriteriumOmschrijving

Max. afw. rel. RT Std (0.125-2.0 µg/l)

Max. afw. ionratio Std (0.25-2.0 µg/l)

Cortisol

Verloop responsfactor

Lineariteit

ShewartkaartJuistheid MMS 0.50 µg/l

Criterium

CriteriumOmschrijving

0.000

0.100

0.200

0.300

0.400

0.500

0.600

0.700

0.800

0.900

1.000

0.00 0.50 1.00 1.50 2.00 2.50

Series1

Series2

Linear (Series1)

APPENDIX 3 FOR SOP-A-1137 CHECKLIST PROCEDURE

o Pipette 2 ml of urine in tube. (tube of 12 ml with screw cap) o Spike the MMS line according the table below and mix o Spike de 1st line control the same as with MMS 0.25 µg/l o Spike de other samples with 20 µl of internal standard solution 0.1 mg/l o Adjust the pH 5.2 ± 0.2 for hydrolysis with 1 ml of acetate buffer 2 M pH 5.2 and adjusted with acetic acid

o Add: 25 µl of β-Glucuronidase/arylsulfatase , mix and hydrolyse during 2 hours at 55°C (water bath) o Add after hydrolysis and cooling down to room temperature, 6 ml of TBME and shake during 30 seconds o Centrifuge for 2 min at 2500 g o Transfer the TBME phase into a clean tube o Repeat the TBME extraction and add the TBME phase to the TBME of the 1st extraction o Evaporate the TBME till it is dry (55°C ± 5°C) o Add 1 ml of acetonitrile, mix and sonicate for 2 min. o Add 3 ml of 0,2 M acetate buffer pH 5.2 and mix o Condition a Strata-X SPE column (60 mg / 3 ml; 8B-S100-UBJ) successively with 3 ml of MeOH and 3 ml of H2O o Transfer the extract to the SPE column o Wash the column successively 2x with 3 ml of MeOH / 2% NH4OH 45:55 (v/v) and then 1x with 3 ml of H2O o Dry the SPE column during 2 min under vacuum o Eluate with 3 ml of MeOH o Evaporate till it is dry (55°C ± 5°C) o Dissolve in 100 µl of H2O / CH3CN + 0.1% acetic acid (75:25 v/v). Mix and sonicate for 2 min o Centrifuge the samples for 5 min at 2500 g o Transfer the end extract into a LC vial o Inject 20 µl Mobile phase A: 0.1% acetic acid in H2O Mobile phase B: 0.1% acetic acid in CH3CN

Preparation MMS

MMS

MSO III (0.025 mg/l) (4.3.5) (µl)

MSO II (0.1 mg/l) (4.3.4)

(µl)

ISO II (0.1 mg/l) (4.3.8)

(µl)

MMS 0.0 µg/l 0 - 20 MMS 0.125 µg/l 10 - 20 MMS 0.25 µg/l 20 - 20 MMS 0.5 µg/l - 10 20 MMS 1.0 µg/l - 20 20 MMS 2.0 µg/l - 40 20

External calibration line: Pipette for each calibration point an amount of (internal) standard solution in a vial of 2 ml according the table below. Evaporate till it is dry and dissolve in water / acetonitrile 75:25 (v/v) according the table below: Standard

µg/l MSO II

(0.1 mg/l) (4.3.4) (µl)

MSO IV Cortisol/cortisone (1.0 mg/l) (4.3.6)

(µl)

ISO II (0.1 mg/l) (4.3.8)

(µl)

After evaporation dissolve in Water/acetonitrile 75:25 (0.1% HAc)

(µl)

0.0 0 - 40 200 0.125 10 - 80 400 0.25 10 - 40 200 0.5 20 - 40 200 1.0 40 - 40 200 2.0 80 - 40 200 5.0 - 20 40 200 10.0 - 40 40 200 25.0 - 100 40 200 50.0 - 200 40 200

APPENDIX 4 FOR SOP-A-1137 EXAMPLE CHROMATOGRAPH MMS 0.5 µg/l

APPENDIX 4 FOR SOP-A-1137 EXAMPLE CHROMATOGRAPH MMS 0.5 µg/l