Moving a Method From GC/NPD to LC/MS - Trace Organic · Moving a Method From GC/NPD to LC/MS Drugs...

April 2008 WCTOWApril 2008 WCTOW 11Ralph Hindle, Vogon Laboratory Ralph Hindle, Vogon Laboratory

Services, Calgary, ABServices, Calgary, AB

Moving a Method From Moving a Method From GC/NPD to LC/MSGC/NPD to LC/MS

Drugs in UrineDrugs in UrineCentre for ToxicologyCentre for ToxicologyUniversity of CalgaryUniversity of Calgary

November 2007November 2007Ralph Hindle, Vogon Laboratory Ralph Hindle, Vogon Laboratory


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November 2007November 2007Ralph Hindle, Vogon Laboratory Ralph Hindle, Vogon Laboratory


Vogon CorporateVogon Corporate

April 2008 WCTOWApril 2008 WCTOWRalph Hindle, Vogon Laboratory Ralph Hindle, Vogon Laboratory

Services, Calgary, ABServices, Calgary, AB 44

HistoryHistory• 6 compounds in method

– Methylphenidate (Ritalin)– Meperidine (Demerol)– Normeperidine– Propoxyphene (Darvon)– Norpropoxyphene– Pentazocine (Talwin)

• Non-reactive to immunoassay screening– GC/NPD Screen– GC/MSD confirmation



Initial ThoughtsInitial Thoughts• Why not just skip NPD analysis?

– Same sample workup as MSD– NPD seems redundant

• Instrument availability– NPD available– MSD used for many methods– Only presumptive positives need to be

confirmed– Addition of Ritalinic acid



IssuesIssues• Screening and confirmation using 3 methods• Want 3 additional compounds• Time consuming

– Sample workup• LLE + centrifuge + 2 transfers + N-evap with solvent

exchange• 7 samples prep time = 45 - 60 minutes

• GC/MSD Confirmation– Poor peak shapes and reproducibility– Recoveries 38 - 132% RSD = 6.6 – 9.5% (n=6)– Confidence in results is low – Injection cycle time: 30 min

April 2008 WCTOW Ralph Hindle, Vogon Laboratory Services, Calgary, AB

7

StructuresMethylphenidate (Ritalin)

Pentazocine

Buprenorphine


8

Propoxyphene Structure



GC/NPD OutlineGC/NPD Outline

• Make urine basic with KOH• Liquid-liquid extraction to diethyl ether• Concentrate with N-evap• Solvent exchange to toluene• GC/NPD with ISTD (diphenylamine)

– Presumptive positives (> 200 ng/mL) -> GC/MS



GC/MS ConfirmationGC/MS Confirmation

• Presumptive positives injected in scan mode– Library match > 80% goes to confirmation

• New sample aliquot– Same sample workup– Labeled drugs as ISTD’s – GC/MSD



Additional CompoundsAdditional Compounds• Buprenorphine and norbuprenorphine

– Current method• Hydrolyzed• SPE cleanup• Derivatization• GC/MSD

• Ritalinic acid– Ritalin metabolite– Not analyzed



Alternative Method 1Alternative Method 1

• LC/MS/MS (Agilent QQQ with ES+ MRM)– 6 current compounds + 3 additional compounds– Recoveries 81-109% SD = 0.4 - 5.2% (n=4)– Quantitation and confirmation in one method– Confidence in results (2 transitions each = 4 EU points)– Very fast

• Sample workup– 1:10 dilution + filtration (sample needs acidification!)– 7 samples prep time = 17 minutes

• Injection cycle time: 9 min



Alternative Method 2Alternative Method 2• LC/MSD (Agilent 1100 with ES+ SIM)

– Recoveries 51 -109% (n=4); pentazocine interference (~ 1,800 ng/mL)– Screening method

• Very low fragmentation to qualifier ions– 6 compounds 2-10% of target– 2 compounds 15-25%– 1 compound at 100%

• Common ions in SIM require chromatographic separation– Confidence in results (1 target ion + 1 qualifier ion = 2 EU points)– Needs confirmation method (GC/MS or LC/MS/MS)– Very fast

• Sample workup– 1:10 dilution + filtration– 7 samples prep time = 17 minutes

• Injection cycle time: 17 min



Acetonitrile Acetonitrile vsvs. Methanol. Methanol



Compound RecoveriesCompound RecoveriesCompound GC/NP/MSD LC/MS/MS LC/MSD

Ritalinic acid 81.4 ± 2.8 51.0 ± 0.7

Methylphenidate 38 / 66 94.0 ± 0.4 90.5 ± 1.6

Meperidine 91 / 110 93.9 ± 1.4 95.4 ± 1.5

Normeperidine 132 / 69 87.6 ± 1.9 77.7 ± 0.8

Pentazocine 114 / 118 93.5 ± 0.9 Interference

Propoxyphene 48 / 100 ± 1.3 105 ± 2.0

Norpropoxyphene 109 ± 1.1 109 ± 1.0

Buprenorphine 99.5 ± 2.2 109 ± 1.6

Norbuprenorphine 95.6 ± 5.2 74.9 ± 1.2



Metabolite AnalysisMetabolite AnalysisRitalinic Ritalinic acidacid

Sample MPH(CFT)

MPH(VLS)

RIT-a(CFT)

RIT-a(VLS)

A 1,280 1,360 - 19,100

B N.D. 3 - 62

C 458 125 - 111,000



Method SummaryMethod Summary

• There are difficulties in all methods– GC/NPD/MSD

• Highly variable• Compounds disappear• Solvent exchange is tedious

– LC/MSD• Low fragmentation gives low qualifier response

– None for ritalinic acid in urine, due to interferences• Will require confirmation method (GC/MSD or LC/MS/MS)• Pentazocine interference

– Can be overcome using 2 qualifiers

– LC/MS/MS• Instrument availability


18

Glucuronic Acid


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StructuresMethylphenidate (Ritalin)

Pentazocine

Buprenorphine



GlucuronidesGlucuronides

• Not all standards available• Add ion for conjugate (+ 176 amu)• Need to hydrolyze

– Quantitate on non-conjugated compounds



Method ComparisonMethod ComparisonCharacteristic GC/NP/MSD LC/MS/MS LC/MSD

Prep time (n = 7) 45 – 60 min 17 min 17 min

Instrument time 30 min 9 min 17 min

Recoveries 38 – 132% 81 – 109% 51 – 109%

CV’s 7 – 9% 0.4 – 5% 0.7 - 2%

Confirmation Points 2-3 4 1-2

Acceptable chromatography

No(peak shapes)

Yes No(pentazocine interference; blanks up to 1,800 ng/mL )

Compound stability No(variable responses)

Yes Yes

LOQ (ng/mL) 100 1 – 10 100

Instrument available Yes No Yes


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Method FlowchartProtein Precipitation

and Dilution

LC/MS SIM

Above cut-off?

New Aliquot

New Aliquot Hydrolyze

Confirmation Method

BUP, norBUP, PEN,

or Gluc’s?

Report “Positive”

Above Cutoff?

Report “Negative”

NO

YES

YES

YES

NO

NO



ValidationValidation• Linearity to 2,500 ng/mL: R2 0.994 - 0.999• LOD’s lower than GC: 10 - 100 ng/mL• Carryover ~ 20 ng/mL after high standard• Precision at cutoff (300 ng/mL):

– Method < 12% (n = 11)– Instrument < 2% (n = 7)

• Recoveries: 73 - 129%• GC - LC comparison: general agreement



Thank You!Thank You!

• WCTOW is a sure sign of spring• April showers bring May flowers…



May 24, 2007May 24, 2007



AcknowledgementsAcknowledgements

• Centre for Toxicology (U. Calgary)– Dr. David Kinniburgh– Lorinda Butlin– Kathy Hunt

Moving a Method From GC/NPD to LC/MS - Trace Organic · Moving a Method From GC/NPD to LC/MS Drugs...

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