Clinical Urine Mega Method by LC-MS/MS

Nadine Koenig1, Crystal Xander1, Melanie Stauffer1, Dean Fritch2
1 Health Network Laboratories, 794 Roble Road, Allentown, PA 18109
2 Analytical Associates, 225 Millwood Drive, East Greenville, PA
Presented at MSACL 2019

Introduction

This improved sample preparation method allows for the quantitative measurement of over 60 drugs of different classes in urine for clinical purposes. Drugs of abuse include naturally occurring, semi-synthetic and synthetic drugs. The use of hydrolysis in the analysis of natural and synthetic drugs in urine has become standard practice in toxicology labs. Many laboratories currently use solid phase extraction or solid liquid extraction techniques in the sample preparation of urine. This method quantitatively measures multiple drugs of different classes in urine for clinical purposes. This method is known as the CLUMM (Clinical Urine Mega Method) and run on the Sciex 4500 using the Phenomenex Phenyl-hexyl Kinetex analytical column. The samples are hydrolyzed, then prepared using a dilute and filter technique followed by LC/MS/MS analysis.

60 Drug Samples Tested

  • Amphetamine
  • Codeine
  • Meperidine
  • Nortriptyline
  • Methamphetamine
  • Morphine
  • Normeperidine
  • Duloxetine
  • MDA
  • 6 MAM
  • Methadone
  • Ketamine
  • MDMA
  • Hydrocodone
  • EDDP
  • Norketamine
  • Gabapentin
  • Hydromorphone
  • Mitragynine
  • Methylphenidate
  • Pregabalin
  • Norhydrocodone
  • 7-Hydroxymitragynine
  • Ritalinic Acid
  • 2-Hydroxyethylflurazepam
  • Dihydrocodeine
  • Tapentadol
  • Zolpidem
  • 7 Aminoclonazepam
  • Oxycodone
  • N-Desmethyl Tapentadol
  • Carboxyzolpidem
  • aOH-Alprazolam
  • Oxymorphone
  • Tramadol
  • THC-COOH
  • Diazepam
  • Noroxycodone
  • O-desmethyltramadol
  • Nicotine
  • Nordiazepam
  • Buprenorphine
  • Carisoprodol
  • Cotinine
  • Oxazepam
  • Norbuprenorphine
  • Meprobamate
  • 3-OH-Cotinine
  • Temazepam
  • Fentanyl
  • Cyclobenzaprine
  • Butalbital
  • aOH-midazolam
  • Norfentanyl
  • Benzoylecgonine
  • Pentobarbital (qualitative only)
  • Lorazepam
  • Acetylfentanyl
  • PCP
  • Phenobarbital (qualitative only)
  • Secobarbital (qualitative only)

Equipment

  • Sciex 4500 LC-MS/MS System
  • Phenomenex Phenyl-hexyl Kinetex analytical 100A 50 x 4.6 mm column
  • Eppendorf Mix Mate
  • Thomson eXtreme Filter Vials, 0.2µm

Sample Preparation

A. Urine Specimens are 1.5mL and are kept refrigerated. Allow standards, specimens and controls to come to room temperature. Turn Block Heater on to 55ºC±2ºC. Label one 1.5 mL Safe-Lock Tube and one Thomson vial for each blank, standard, control and client specimen. For samples falling outside the calibration range, make appropriate dilutions using Negative Urine and record on the run sheet. The goal is to prevent mass spectral distortion (failing ion ratios) that occurs in a sample that is too concentrated while keeping the concentration of the diluted sample above the cutoff (or a least the limit of quantitation).

NOTE: The maximum dilution allowed for this analysis is 1:20. This dilution is for all analytes with the exception of THC. Perform this dilution in a separate 12x75 mm glass tube. Place 950 µL of Negative Urine into the tube using the 200-1000 µL and add 50µL of sample requiring dilution into the same tube. Vortex for 20-30 seconds.

For the LC Check, place 400 µL of 2% Methanol into a 12 x 75 mm glass culture tube. Add 20 µL of working IS and 1 µL of Cutoff Calibrator Spiking Standard A and 1 µL of Cutoff Calibrator Spiking Standard B. Vortex and transfer to an autosampler vial with insert. To each 1.5 mL Safe-Lock Tube add 90 µL of Rapid Hydrolysis Mixture. Prepare 1.5 mL Safe-Lock Tubes for analysis according to table 1. Cap and vortex for 5 minutes at 850 rpm using the Eppendorf Mix Mate. Incubate at 55ºC±2ºC for 30 minutes uncapped. Allow tubes to come to room temperature.

Add 200 µL of 2% Methanol to each Thomson Vial. Give each Eppendorf tube a quick vortex and add 200 µL of the hydrolyzed urine sample to its respective Thomson Vial. Place Thomson Filter Plunger on top of Thomson Vial. Press filter plunger down approximately ¼ of the way into each of the Thomson Vials. Vortex for 5 minutes at 1750 rpm using the Eppendorf Mix Mate.

B. Add 200 µL of 2% Methanol to each Thomson Vial. Briefly vortex each sample tube. 200 µL of the hydrolyzed urine sample should be added to its respective Thomson Vial. Place Thomson Filter Plunger on top of Thomson Vial. Press filter plunger down approximately ¼ of the way into each of the Thomson Vials. Vortex for 5 minutes at 1750 rpm using the Eppendorf Mix Mate.

Table.1
TUBEAMOUNT OF CLINICAL/FUMM2 WORKINGI.S.(µL)AMOUNT OF CUTOFF CALIBRATOR SPIKING STANDARD A(µL)AMOUNT OF CUTOFF CALIBRATOR SPIKING STANDARD B(µL)AMOUNT OF QUALITY CONTROL Spiking Standard A(µL)AMOUNT OF QUALITY CONTROL Spiking Standard B(µL)AMOUNT OF NEGATIVE URINE(µL)AMOUNT OF SPECIMEN(µL)
Blank20----200-
Level 12011--198-
Level 22022--196-
Level 32055--190-
Level 4202020--160-
Level 5205050--100-
Low Control20--22196-
High Control20--3030140-
Specimens20-----200
LOD (when applicable)200.50.5--199-

Results

Final concentrations (ng/mL) including linearity for the various analytes including controls can be found in Table 2.

Validation of any method must include evaluation of interfering substances/co-eluting peaks. There may be unknown substances in certain specimens which co-elute with the analyte or the internal standard and may cause low recovery or cause ion ratios to fail. Seven analyte mixes, see Fig. 1 were evaluated for interference. Fig. 2 shows the list of analytes that show interference with one or more analytes in the above mixes. The analytes in Fig. 2 had % accuracies exceeding 60-140% when spiked into the low control. There are unknown substances that interfere with Barbiturates*. Examples of mass spectrum of some of the analytes can be seen in Fig. 3-10.

*Note: If any of these analytes appears positive in any patient sample they will be reflexed and repeated by an appropriate alternate method.

Table.2Concentrations of the various analytes
AnalyteLevel 1
(LOD/LOQ/CUTOFF CONCENTRATION)
Level 2Level 3Level 4Level 5
(LINEARITY)
Low ControlHigh Control
Amphetamine100200500200050002003000
Methamphetamine100200500200050002003000
MDA100200500200050002003000
MDMA100200500200050002003000
Gabapentin500100025001000025000100015000
Pregabalin500100025001000025000100015000
2-Hydroxyethylflurazepam75150375150037501502250
7-Aminoclonazepam75150375150037501502250
aOH-Alprazolam75150375150037501502250
Diazepam75150375150037501502250
Nordiazepam75150375150037501502250
Oxazepam75150375150037501502250
Temazepam75150375150037501502250
aOH-midazolam75150375150037501502250
Lorazepam75150375150037501502250
Codeine50100250100025001001500
Morphine50100250100025001001500
6-MAM5102510025010150
Hydrocodone50100250100025001001500
Hydromorphone50100250100025001001500
Norhydrocodone50100250100025001001500
Dihydrocodeine50100250100025001001500
Oxycodone50100250100025001001500
Oxymorphone50100250100025001001500
Noroxycodone50100250100025001001500
Buprenorphine5102510025010150
Norbuprenorphine5102510025010150
Fentanyl1252050230
Norfentanyl1252050230
Acetylfentanyl1252050230
Meperidine50100250100025001001500
Normeperidine50100250100025001001500
Methadone100200500200050002003000
EDDP100200500200050002003000
Mitragynine10205020050020300
7-Hydroxymitragynine10205020050020300
Tapentadol100200500200050002003000
N-Desmethyl Tapentadol100200500200050002003000
Tramadol100200500200050002003000
O-desmethyltramadol100200500200050002003000
Carisoprodol100200500200050002003000
Meprobamate100200500200050002003000
Cyclobenzaprine50100250100025001001500
Benzoylecgonine100200500200050002003000
PCP2550125500125050750
Nortriptyline50100250100025001001500
Duloxetine50100250100025001001500
Ketamine100200500200050002003000
Norketamine100200500200050002003000
Methylphenidate10205020050020300
Ritalinic Acid100200500200050002003000
Zolpidem75150375150037501502250
Carboxyzolpidem2550125500125050750
Butalbital20040010004000100004006000
Pentobarbital20040010004000100004006000
Phenobarbital20040010004000100004006000
Secobarbital20040010004000100004003000
THC-COOH15307530075030450
Nicotine100 (LOD/LOQ)200(Cutoff Conc)500200050002003000
Cotinine100 (LOD/LOQ)200 (Cutoff Conc)500200050002003000
3-trans-OH-Cotinine100 (LOD/LOQ)200 (Cutoff Conc)500200050002003000

Analyte mixtures that were used to evaluate interfering substances

Mix 1

  • Acetaminophen
  • Caffeine
  • Cotinine
  • Ibuprofen
  • Naproxen
  • Nicotine
  • Phentermine
  • Pseudoephedrine

Mix 2

  • Gabapentin
  • Pregabalin
  • Salicylic acid
  • Valproic Acid
  • Vigabatrin

Mix 3

  • Aripiprazole
  • Lacosamide
  • Oxcarbazepine
  • Propoxyphene
  • Rufinamide
  • Warfarin

Mix 4

  • Alprazolam
  • Fluconazole
  • Cimetidine
  • Hydrochlorothiazide
  • Citalopram
  • Lamotrigine
  • Clopidogrel bisulfate
  • Levothyroxine
  • Clonazepam
  • Methylphenidate
  • Dextromethorphan
  • Omeprazole

Mix 5

  • Carbamazepine
  • Phenytoin
  • Levetiracetam
  • Sertraline
  • Metformin
  • Topiramate
  • Phenobarbital
  • Zolpidem Tartrate
  • Phenylephrine
  • Zonisamide

Mix 6

  • Amlodipine besylate
  • Atorvastatin Calcium Salt
  • Azithromycin
  • Bupivacaine HCL
  • Cetirizine diHCL
  • Dimenhydrinate
  • Lisinopril
  • Loratidine

Mix 7

  • Montelukast Sodium
  • Pioglitazone HCl
  • Prednisolone
  • Prednisone
  • Procainamide HCl
  • Simvastatin

Analytes that interfered with other analytes

Interferring Peaks

  • Acetylfentanyl
  • Cotinine
  • Gabapentin
  • Methylphenidate
  • Mitraginine
  • Nicotine
  • o-desmethyl tramadol
  • OH-mitraginine
  • Phenobarb
  • Pregabalin
  • Ritalinic acid
  • Tapentadol
  • Temazepam
  • Tramadol
  • Zolpidem
  • 3-OH-cotinine
APP Clinical Urine Mega Method 001

fig.3Buprenorphine

APP Clinical Urine Mega Method 002

fig.4Carisoprodol

APP Clinical Urine Mega Method 003

fig.5Fentanyl

APP Clinical Urine Mega Method 004

fig.6Meprobamate

APP Clinical Urine Mega Method 005

fig.7Norbuprenorphine

APP Clinical Urine Mega Method 006

fig.8Norfentanyl

APP Clinical Urine Mega Method 007

fig.9O-desmethyltramadol

APP Clinical Urine Mega Method 008

fig.1Tramadol

Conclusion

This method quantitatively measures multiple drugs of different classes in urine for clinical purposes. This method is known as the CLUMM (Clinical Urine Mega Method). This new improved method allows for a large sample panel, reduces sample prep time, limits transfer steps, improves column life, and reduces instrument downtime.

Title

Clinical Urine Mega Method by LC-MS/MS

Description

This improved sample preparation method allows for the quantitative measurement of over 60 drugs of different classes in urine for clinical purposes. Drugs of abuse include naturally occurring, semi-synthetic and synthetic drugs. The use of hydrolysis in the analysis of natural and synthetic drugs in urine has become standard practice in toxicolog...

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