OPIATES in Urine by GC-MS – Code GC45010 (Morphine, Dihydrocodeine, Codeine, Ethylmorphine, 6-Monoacethylmorphine) Method of Confirmation by GC-MS

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OPIATES in urine by GC-MS – Code GC45010 (Morphine, Dihydrocodeine, Codeine, Ethylmorphine, 6-Monoacethylmorphine) Method of Confirmation by GC-MS

INTRODUCTION

Heroin (diacetylmorphine) is a semisynthetic drugs of abuse obtained from morphine, a major opium alkaloids, by reaction with acetic anhydride. Inside the body, heroin is rapidly hydrolysed to 6monoacetylmorphine and then to morphine. It is then conjugated with glucuronic acid in position 3 and 6 of the morphine molecule forming three six glucuronide. The latter two metabolites are excreted in urine together with a small percentage of free morphine. We can also find small amounts of codeine, morphine, opium, and acetylcodeine which is obtained by acetylation of codeine during the preparation of heroin. The methods of screening measure, through an antigenantibody reaction, the opioid class of substances. In the event of a positive screening (cutoff > or = 300 ng/ml) analysis is confirmed, usually with gaseous liquid chromatography coupled with a mass spectrometry detector. This method enables the total morphine urine (after hydrolysis of glucuronide), codeine and 6monoacetylmorphine may be present in small quantities after extraction of these substances from the urine matrix in micro solid phase extraction columns. The method also allows the determination of dihydrocodeine and of etylmorphine. These two substances are opioids, which provide a positive result with screening methods for this class of opiates, are in fact the active ingredients of certain anticough products (eg, dihydrocodeine is the active ingredient in paracodine) or found in association with other active ingredients in pharmaceutical preparations with analgesicantipyretic (ei. Mindolmerk etylmorphine with propifenazone and contains caffeine). It is important that the method of analysis can identify these two substances and differentiate them from other molecules above.

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Release N° 013 Opiates in urine by GC/MS May 2013

1 TECHNICAL FEATURES

Principle of the Method :

This method allows to determinate the Morphine in urine (the main metabolite of Heroin ), the Dihydrocodeine, the Codeine, the Ethylmorphine and the 6-Monoacethylmorphine after hydrolysis of urines and SPE extraction and reading in GCMS.

Recovery: > 95 %

Sensitivity: < 10,0 ng/ml

Dynamic Range: 10 – 10.000 ng/ml

LOD: 2 ng/ml

LOQ: 6 ng/ml

Cut-off: Morphine 100 ng/ml Dihydrocodeine 100 ng/ml Codeine 100 ng/ml Ethylmorphine 100 ng/ml 6-Monoacethylmorphine 100 ng/ml Ref. Procedure applicative del procedimento n.99/cu 30 ottobre 2007 Components of the kit : All the components are readytouse. Stability: 3 years at 2 – 8 °C.

Reagent A – Hydrolysis Enzyme, 1 x 1 ml To reconstitute with 9,0 ml of Reagent B. After reconstitution, the enzyme is stable 1 year at 2–8 °C. Reagent B – Buffer Solution for hydrolysis, 1 x 15 ml

Reagent C – Conditioning Solution N° 1 and Wash Solution N° 3, 1 x 250 ml

Reagent D – Conditioning Solution N° 2, 1 x 100 ml

Reagent E – Buffer Solution, 1 x 100 ml

Reagent F – Wash Solution N° 1, 1 x 100 ml

Reagent G – Wash Solution N° 2, 1 x 100 ml

Reagent H – Eluting Solution, 1 x 50 ml

Reagent I – Diluting Solution, 1 x 25 ml

Reagent N – Test Solution, 1 x 0,5 ml See Warnings

Reagent S – Internal Standard Solution, 1 x 300 µl To dilute 1:10 with H 2O before use

Urine Calibrator lyophil. – Level 1, 2 x 5 ml Code CC45016 (packed separately – see data sheets) Urine Calibrator lyophil. – Level 2, 2 x 5 ml Code CC45016 (packed separately – see data sheets) Urine Calibrator lyophil. – Level 3, 2 x 5 ml Code CC45016 (packed separately – see data sheets) Urine Calibrator lyophil. – Level 4, 2 x 5 ml Code CC45016 (packed separately – see data sheets) Urine Calibrator lyophil. – Level 5 , 2 x 5 ml Code CC45016 (packed separately –

2 see data sheets) Urine Calibrator lyophil. 6-MAM – Level 1, 2 x 5 ml Code CC45116 (packed separately – see data sheets) Urine Calibrator lyophil. 6-MAM – Level 2, 2 x 5 ml Code CC45116 (packed separately – see data sheets) Urine Calibrator lyophil. 6-MAM – Level 3, 2 x 5 ml Code CC45116 (packed separately – see data sheets) Urine Calibrator lyophil. 6-MAM – Level 4, 2 x 5 ml Code CC45116 (packed separately – see data sheets) Urine Calibrator lyophil. 6-MAM – Level 5, 2 x 5 ml Code CC45116 (packed separately – see data sheets) Reagent M – Derivatization Solution, 3 x 1 ml (3 ampullas of 1 ml) See Warnings

Clean-up Columns, 50 pcs

Minimum Instrumental equipment required: GCMS Instrument Operational Computer

Optional Equipment: Autosampler

Urine Collection Procedure: Take 10 ml of urine If stored at 20 °C, the sample is stable about 3 months.

Release N° 013 Opiates in urine by GC/MS May 2013

PREANALYTICAL PROCEDURE:

STEP 1 : In a glasstest tube with teflon cap pipette :

• 450 l of Reagent I – Diluting Solution • 50 l of Reagent N – Test Solution Vortex for 20 seconds

STEP 2 : Dry the eluate by the help of an helium or nitrogen flow (at this step the work temperature must be < 40 °C).

STEP 3 : Put in 50 l of Reagent M Derivatization Solution

Vortex for 20 seconds

STEP 4 : Incubate at 70 °C for 20 minutes Cooling at Room Temperature

STEP 5 : Transfer the whole sample in vials goblet form

NOTE : Before starting the analytical session it would be better to inject 1 l of this solution in GC to identify the time of retention of Opiates, that must be similar to that shown in below Fig. If the Test has given positive result, proceed with the analytical session. If it wouldn’t be so, verify the functionality of the analytical system .

ANALYTICAL PROCEDURE:

STEP 1 : Enzyme Reconstitution and Internal Standard dilution

Add at Reagent A – Hydrolysis Enzyme (containing 1 ml) 9 ml of Reagent B – Buffer Solution for hydrolysis.

Mix by inversion

N.B: Dilute the Reagent S – Internal Standard Solution 1:10 with H 2O, preparing the sufficient sample size to cover the assay.

N.B.: this dilution is prepared fresh for each run

Release N° 013 Opiates in urine by GC/MS May 2013

STEP 2 : Samples Enzymatic Hydrolysis

Make a Calibration Curve on 5 points:

Calibrator Calibrator Calibrator Calibrator Calibrator Sample Controls Opiates + Opiates + Opiates + Opiates + Opiates + + 6MAM 6MAM 6MAM 6MAM 6MAM Controls Level 1 Level 2 Level 3 Level 4 Level 5 6-MAM Urine Calibrator 500 l Opiates – Level 1 Urine Calibrator 500 l 6MAM – Level 1 Urine Calibrator 500 l Opiates – Level 2 Urine Calibrator 500 l 6MAM – Level 2 Urine Calibrator 500 l Opiates – Level 3 Urine Calibrator 500 l 6MAM – Level 3 Urine Calibrator 500 l Opiates – Level 4 Urine Calibrator 500 l 6MAM – Level 4 Urine Calibrator 500 l Opiates – Level 5 Urine Calibrator 500 l 6MAM – Level 5 Sample 1,0 ml Controls 500 l Controls 6 -MAM 500 l Reagent S – 20 l 20 l 20 l 20 l 20 l 20 l 20 l Internal Std Sol. (diluted 1:10 with HPLC grade H2O) Reagent A – 200 l 200 l 200 l 200 l 200 l 200 l 200 l Enzyme reconstituted Control the pH is ≅ 5,0 – In case, correct it

N.B: The values of concentration of the Cocaine, Morphine, Dihydrocodeine, Ethylmorphine and 6- MAM calibrators mixed together are the HALF to that shown in the table of warnings!!!! In vortex for 20 seconds

Release N° 013 Opiates in urine by GC/MS May 2013

STEP 3 : Incubate at 50 °C for 3 hours.

STEP 4 : Conditioning of the Cleanup columns

• Pour out in column 1,5 ml of Reagent C – Conditioning Solution N° 1 and Wash Solution N° 3 Reject the eluate • Pour out in column 2 ml of Reagent D – Conditioning Solution 2 Reject the eluate

STEP 5 : Preparation of the sample

Dispense in test tube :

Hydrolysed Hydrolysed Hydrolysed Hydrolysed Hydrolysed Sample Controls Calibrator – Calibrator – Calibrator Calibrator – Calibrator – Level 1 Level 2 – Level 3 Level 4 Level 5 Hydrolysed 1200 l Calibrator – Level 1 Hydrolysed 1200 l Calibrator – Level 2 Hydrolysed 1200 l Calibrator – Level 3 Hydrolysed 1200 l Calibrator – Level 4 Hydrolysed 1200 l Calibrator – Level 5 Hydrolysed 1200 l Sample Hydrolysed 1200 l Controls Reagent E – 2000 l 2000 l 2000 l 2000 l 2000 l 2000 l 2000 l Buffer Solution

Vortex for 20 seconds

STEP 6 : Pour the whole sample and the Calibrator, previously prepared, in the Cleanup columns and discharge in a flow of 1 ml/ min (droop by drop).

STEP 7 : Cleanup columns Wash:

• Wash with 2 ml of Reagent F – Wash Solution 1 Discharge the percolated liquid • Wash with 2 ml of Reagent G – Wash Solution 2 Discharge the percolated liquid • Wash with 2 ml of Reagent C – Conditioning Solution 1 and Wash Solution 3 Discharge the percolated liquid and dry with a light flux of air

Release N° 013 Opiates in urine by GC/MS May 2013

STEP 8 : Elution

• Elute with 1 ml of Reagent H – Eluting Solution

Gather the eluate in glass test tubes and vortex

STEP 9 : Dry the eluate by the help of an helium or nitrogen flow (at this step the work temperature must be < 40 °C).

STEP 10 : Take again 50 l of Reagent M Derivatization Solution

Vortex for 20 seconds

STEP 11 : Incubate at 70 °C for 20 minutes

Cooling at Room Temperature

STEP 12 : Transfer the whole sample in vials goblet form

INJECTION :

• Inject 1 l of the solution in the GC

Release N° 013 Opiates in urine by GC/MS May 2013

7 OPIATES in urine - Warnings

REAGENT N: TEST SOLUTION

Morphine about 10.000 ng/ml Dihydrocodeine about 10.000 ng/ml Codeine about 10.000 ng/ml Ethylmorphine about 10.000 ng/ml 6-Monoacethylmorphine about 10.000 ng/ml

REAGENT M: DERIVATIZATION SOLUTION

Once opened an ampulla, transfer the whole content, 1 ml, in a glass vial. Close carefully the cap and store at Room Temperature.

SET OF GAS-CROMATOGRAPH:

• VF5ms Column 30 m x 0,25 mm, 0,25 m (conditioned) • Injector’s Temperature 280 °C • Split Report Start: ON split 20 0 min: OFF 1 min: ON split 100 3 min: ON split 20 • Temperature 120 °C x 3,25 minutes + 20 °C/min up to 250 °C x 1 minute + 40 °C/min up to 300 °C x 3 minutes (run 15 minutes) • Helium Gas 1 ml/min SET OF MASSA DETECTOR (4000) :

• Range of Massa 60 – 500 • Temperature of Massa: 180 °C; • Temperature Transfer Line: 270 °C; • Temperature Manifold: 80 °C • Filament on: 5 minutes

CONDITIONING OF VF-5 MS COLUMN and SYRINGE

Before use wash the syringe with ACETONE. Follow the manufacturer prescription. Do not condition column/s if connected to the Massa Detector. CLEANING OF THE COLUMN

Disconnect the detector. Keep the column at the highest temperature for the recommended time. (See the manufacturer ’s instructions)

ACCESSORIES AND CONSUMABLES

CODE DESCRIPTION PACKAGING CC45016 Calibrator in urine for Opiates 2 x 5 x 5 ml CC45017 Control in urine for Opiates – Level 1 6 x 5 ml CC45018 Control in urine for Opiates – Level 2 6 x 5 ml CC45019 Control in urine for Opiates – Levels 1 and 2 2 x 6 x 5 ml CC45116 Calibrator in urine for 6MAM 2 x 5 x 5 ml CC48017 Control in urine for EDDP, 6MAM and Cocaine – Level 1 6 x 5 ml CC48018 Control in urine for EDDP, 6MAM and Cocaine – Level 2 6 x 5 ml Control in urine for EDDP, 6MAM and Cocaine – Levels 1 CC48019 2 x 6 x 5 ml and 2 ZRE13623 RxiSil 5MS (30 m x 0,25 mm) ID, DF=0,25 Capillary Column 1 Pc SCP8944 VF5ms (30 m x 0,25 mm) ID DF=0,25 Column 1 Pc S29057U Standard glass vial of 2 ml with screw cap 1 x 100 Pcs S29046U Caps for Standard glass vials of 2 ml with screw cap 1 x 100 Pcs S24717 Insert 100 ul for vials of 2 ml 1 x 100 Pcs

Opiates in urine (Reference Chromatograms/Spectrum GC-MS)

kCounts CODEINATMS 19 c2 105ng2_10262010.sms Ions: 371.0 Filtered CODEINATMS 600 Ionization Off 60:500 BP 371.0 (999=100%) 8 in EUREKA 371.0 999 500 100%

400

300

200

100 234.0 CO DEINATM12.759 m in S 749 75% 0 kCounts CODEINATMS 21 c3 210ng2_10262010.sms Ions: 371.0 Filtered Ionization Off 60:500 600 178.0 500 585 400 146.0 520 300 50% 196.0 229.0 459 456 200 372.0 CODEIN12.757 ATM m in S 179.0 410 100 380 343.0 342 0 73.0 kCounts CODEINATMS 22 c4 420ng2_10262010.sms Ions: 371.0 Filtered 305 Ionization Off 60:500 313.0 280.0 253 600 25% 240 370.0 214.0 221 500 198 235.0 281.0 94.0 180 173 314.0 165.0 400 147 197.0 154 356.0

C ODEINATMS12.764 m in 134 115.0 126 236.0 126 75.0 300 103 181.0 216.0 95 298.0 83 129.0 158.0 103.0 74 69 76 200 55 58 56

100 0% 0 2.5 5.0 7.5 10.0 12.5 100 200 300 400 minutes m/z Fig. 1 : Urine Sample Codeine Fig. 2 : Massa Spectrum Codeine R.T. 12.76 CODEINE MOLECULAR IONS: 371 – 234 343

9 Opiates in urine (Reference Chromatograms/Spectrum GC-MS)

MCounts Dihydrocodeine TMS derivative 19 c2 105ng2_10262010.sms Ions: 373.0+146.0+282.0 Filtered Dihydrocodeine TMS derivative Ionization Off 60:500 BP 373.0 (999=100%) 159067 in MAINLIB C21H31NO3Si, MW 373 2.5 373.0 70% 999 2.0

1.5 N 1.0 60%

0.5 Dihydro12.438 codeine m in TM S derivative 0.0 MCounts Dihydrocodeine TMS derivative 21 c3 210ng2_10262010.sms Ions: 373.0+146.0+282.0 Filtered 50% Ionization Off 60:500 2.5 O O Si 2.0 O 40% 1.5

1.0

0.5 Dihydrocodeine12.437 m in TM S derivative 30% 374.0 278 0.0 MCounts Dihydrocodeine TMS derivative 22 c4 420ng2_10262010.sms Ions: 373.0+146.0+282.0 Filtered 236.0 Ionization Off 60:500 216 2.5 20% 146.0 179 2.0 282.0 372.0 137 315.0 136 127 1.5 178.0 283.0 10% 90 242.0 316.0 358.0 375.0 1.0 Dihydrocodeine12.442 m in TM S derivative 75 78 297.0 226.0 256.0 69 70 70 129.0 243.0 60 47 53 0.5 39 41

0.0 0% 2.5 5.0 7.5 10.0 12.5 150 200 250 300 350 minutes m/z

Fig. 3 : Urine Sample Dihydrocodeine Fig. 4 : Massa Spectrum Dihydrocodeine

R.T. 12.44 DIHYDROCODEINE MOLECULAR IONS: 373 – 146 282

10 Opiates in urine (Reference Chromatograms/Spectrum GC-MS)

MCounts ETILMORFINATMS 19 c2 105ng2_10262010.sms Ions: 385.0 Filtered ETILMORFINATMS 1.00 Ionization Off 60:500 BP 73.0 (999=100%) 17 in EUREKA

100% 0.75

0.50 385.0 852

0.25 ETILM12.910 ORFINATM m in S 75% 0.00 146.0 696 MCounts ETILMORFINATMS 21 c3 210ng2_10262010.sms Ions: 385.0 Filtered Ionization Off 60:500 1.00

0.75 234.0 192.0 540 519 0.50 50%

0.25 196.0 243.0 ETILM12.911 ORFIN m in ATM S 395 392 193.0 0.00 340 386.0 MCounts ETILMORFINATMS 22 c4 420ng2_10262010.sms Ions: 385.0 Filtered 357.0 294 1.00 Ionization Off 60:500 267 25% 328.0 0.75 204 235.0 280.0 129.0 384.0 152.0 150 154 138 214.0 253.0 137 0.50 131.0 123 179.0 503.0 105 106 296.0 341.0 ETILM12.915 ORFINATM m in S 370.0 91 90 227.0 88 429.0 96 153.0 271.0 78 75 63 56 63 0.25 52

0% 0.00 2.5 5.0 7.5 10.0 12.5 200 300 400 500 minutes m/z

Fig. 5 : Urine Sample Ethylmorphine Fig. 6 : Massa Spectrum Ethylmorphine R.T. 12.91 ETHYLMORPHINE MOLECULAR IONS: 385 – 146 73

Opiates in urine (Reference Chromatograms/Spectrum GC-MS)

kCounts MORFINATMS 19 c2 105ng2_10262010.sms Ions: 429.0 Filtered MORFINATMS 600 Ionization Off 60:500 BP 429.0 (999=100%) 4 in EUREKA 429.0 999 500 100%

400

300

200

100 M OR12.949 FINA TMS m in

0 75% kCounts MORFINATMS 21 c3 210ng2_10262010.sms Ions: 429.0 Filtered Ionization Off 60:500 600

500 234.0 571 400

300 50%

200 M ORFINA12.951 TMm in S 430.0 287.0 100 383 370 324.0 347 414.0 0 330 kCounts MORFINATMS 22 c4 420ng2_10262010.sms Ions: 429.0 Filtered Ionization Off 60:500 196.0 600 251 25% 415.0 500 73.0 237.0 199 172 146.0 173 400 151 221.0 M ORFIN12.954 ATM m in S 288.0 431.0 114 340.0 111 300 94.0 104 371.0 253.0 92 84 403.0 75 69 200 64

100 0% 0 2.5 5.0 7.5 10.0 12.5 100 200 300 400 500 minutes m/z

Fig. 7 : Urine Sample Morphine Fig. 8 : Massa Spectrum Morphine R.T. 12.95 MORPHINE MOLECULAR IONS: 429 – 414 234

Opiates in urine (Reference Chromatograms/Spectrum GC-MS)

MCounts 6Acetylmorphine, 3Otrimethylsilyl 11 c2 105ng2.sms Ions: 399.0+340.0+288.0 Filtered 6Acetylmorphine, 3Otrimethylsilyl Ionization Off 60:500 BP 399.0 (999=100%) 160399 in MAINLIB C22H29NO4Si, MW 399 399.0 999 1.5 100% O Si 1.0

0.5

6Acetylm13.325 orphine,m in 3O trim ethylsilyl O 75% N 0.0 MCounts 6Acetylmorphine, 3Otrimethylsilyl 16 c3 210ng3.sms Ions: 399.0+340.0+288.0 Filtered O Ionization Off 60:500 340.0 1.5 614

O 1.0 50%

0.5 6Acetylm13.329 orph m in ine, 3O trim ethylsilyl

287.0 350 0.0 400.0 MCounts 6Acetylmorphine, 3Otrimethylsilyl 17 c4 420ng3.sms Ions: 399.0+340.0+288.0 Filtered 73.0 303 Ionization Off 60:500 285

25% 342.0 1.5 204.0 201 43.0 173 282.0 139

6Acetylm13.327 orphine,m in 3O trim ethylsilyl 324.0 1.0 42.0 75.0 126 124.0 162.0 105 401.0 94 104 267.0 70.0 83 82 216.0 246.0 356.0 85 58 64 57 68 66 0.5

0% 0.0 2.5 5.0 7.5 10.0 12.5 0 100 200 300 400 minutes m/z

Fig. 9 : Urine Sample 6-Monoacetylmorphine Fig. 10 : Massa Spectrum 6-Monoacetylmorphine R.T. 13.32 6MONOACETYLMORPHINE MOLECULAR IONS: 399 – 340 288

Opiates in urine (Reference Chromatograms/Spectrum GC-MS)

Nalorphine, o,o'bis(trimethylsilyl) MCounts Nalorphine, o,o'bis(trimethylsilyl) 11 c2 105ng2.sms Ions: 455.0+440.0+414.0 Filtered BP 455.0 (999=100%) 161816 in MAINLIB C25H39NO3Si2, MW 457 Ionization Off 60:500 1.00 455.0 999 100% 73.0 0.75 949

Si 0.50

Nalorphine,13.534 m o,o'bis(trimethylsilyl) in O

0.25

75% 0.00 O N MCounts Nalorphine, o,o'bis(trimethylsilyl) 16 c3 210ng3.sms Ions: 455.0+440.0+414.0 Filtered Ionization Off 60:500 1.00

0.75 414.0 O 524 Si

0.50 N alorphine,13.53 8 m o,o'bis(trim in ethylsilyl) 50%

440.0 0.25 407

324.0 0.00 260.0 324 MCounts Nalorphine, o,o'bis(trimethylsilyl) 17 c4 420ng3.sms Ions: 455.0+440.0+414.0 Filtered 308 Ionization Off 60:500 313.0 259 1.00 25% 222.0 415.0 350.0 181 183 441.0 0.75 162 75.0 150

Nalorphine,13.535 m o,o'bis(trim in ethylsilyl) 193.0 372.0 45.0 131 218.0 454.0 74.0 111 266.0 325.0 112 105 0.50 101 97 416.0 85 179.0 84 92 382.0 442.0 122.0 229.0 69 53 63 53 64 56 0.25 0% 0.00 2.5 5.0 7.5 10.0 12.5 100 200 300 400 500 minutes m/z

Fi g. 11 : Urine Samples with Internal Standard Fig. 12 : Massa Spectrum Internal Standard R.T. 13.52 INTERNAL STANDARD MOLECULAR IONS: 455 – 440 414