f - Oxford Journals

16 downloads 0 Views 151KB Size Report
by the Adulterant "Klear" in the GC-MS Analysis of 11-nor-Ag-THC-9-COOH. To the Editor: Gas chromatographic-mass spectrometric (GC-MS) confirmation forĀ ...
Journal

of Analytical

Toxicology,

Vol. 21, May/June 1997

[ Letter to the Editor

A Procedureto Overcome InterferencesCaused by the Adulterant "Klear" in the GC-MS Analysis of 11-nor-Ag-THC-9-COOH To the Editor:

Gas chromatographic-mass spectrometric (GC-MS) confirmation for drugs of abuse is a critical part of the testing process for the identification of drug metabolites in urine. Recently, we observed an increased incidence in the lack of confirmation for 11-nor-M-THC-9-COOH Ion 3 7 7 . 0 0 a m u . f r o m T0011201012.d Ion 371.00 a m u . f r o m T0011201012.d (THCCOOH) by GC-MS of Abundance Abundance specimens screened positive 1200 1200 for cannabinoids by 1000 80O immunoassays. The reason 80O 60O for the lack of confirmation 40O 400 was the inability to recover 20O the internal standard or the 0 -~ , ' I ' ' ' I ' ' ' I ' ' ' I ' ' ' 0 ' ' ' I-r'-I''v |-' ' ' I ' ~ r'-'i i ; drug. Initially, our thoughts 13.8 14.0 14.2 14.4 13.6 13.8 14.0 14.2 14.4 13.6 "Rme (min) Time (min) were that this was probably due to ingestion of some acidic drug such as ibuprofen Ion 473.00 ainu. f r o m T0011201012.d Ion 4 7 6 . 0 0 ainu. f r o m T0011201012.d Abundance Abundance which, when excreted in the 500 -I urine in large amounts, 50o 400 -I consumes the derivatizing 400 i 300 L reagent (1). ~/~_.,,. -,1,.J-, "'-/ ~'"1"%.~-..&~v~._, 300 ": 200 Repeating the analysis with 200 100 ":: a higher level of derivatizing 100 reagent usually corrects the 0- I ' ' - " ~ - - ' F - , , " I--', ' ' I ' ' ' I ' ' ' 0--, , , I ' ' '--1 --'~ ' ~ I ' r'-"r'-I ' ' r - 13.8 14.0 14.2 14.4 13.6 13.8 14.0 14.2 14.4 13.6 problem, and confirmation is rime (min) 13me (min) accomplished when the interference is caused by Ion 4 8 8 . 0 0 a m u . f r o m T0011201012,d Ion 4 9 4 . 0 0 a m u . f r o m T0011201012.d acidic drugs such as Abundance Abundance 400 ibuprofen. This was not the 1200 -i /~./ case for many of the 300 ~--,~_~NV~/'~ -~.,-,A~,~-,-~,\/~,.'kN',.,. situations we encountered. 8oo-i /~/ 200 One of the products currently 100 advertised as being able to mask the detection of 0 o-,-._77, ,--,'.; , . , ,,,, ,13.6 13.8 14.0 14.2 14.4 13.8 14.0 14.2 14.4 13,6 marijuana use is sold under "13me (rain) Time (min) the name "Klear". Therefore, we acquired the product to Figure 1. GC-MS analysisof a urine specimencontaining50 ng/mLTHCCOOH spikedwith 2.5 mg/mL Klear.Ions monitored were as follows: m/z 371,473, and 488 for THCCOOH and 377, 476, and 494 for internal standard. determine if it was the culprit in these situations. Klear is a

\~.~/-~"~,

f\

/'\

,~176

240

)\

Reproduction

(photocopying)

of editorial content of this journal is prohibited without publisher's permission.

Journal of Analytical Toxicology, Vol. 21, May/June 1997

white granular product that is freely soluble in water and nonflammable9It has a melting point in excess of 300~ which suggests that it is an inorganic salt. The UVabsorbance spectrum showed peaks at 211, 298, and 354 nm. An aqueous solution of the product produced a positive potassium iodide starch test, which suggests that it is a nitrite salt. A sample of potassium nitrite was acquired and compared with the product in all the previously mentioned physical characteristics and was found to be identical9In addition, a 1:1 mixture of Klear and KNO2(melting point 355~ with decomposition) melted at > 300~ with no depression in the melting point, which supported the belief that Klear is pure KNO2. Klear and potassium nitrite were then tested separately to check the effect on the recovery of THCCOOHfrom urine specimens. Neither product had significant effect on the results of the immunoassays. However,both resulted in lack of recovery of THCCOOHand its internal standard (THCCOOH-d6).At a 2.5 mg/mL concentration of Klear or potassium nitrite, there was more than 90% loss in the recovery of THCCOOHand the internal standard, and, at higher concentrations (approximately ] 0 mg/mL), there was no recovery. Having identified the cause of the interference, our goal was to develop a method to overcome the problem. After the addition of the internal standard to the urine specimen, approximately 250 mg of sodium bisulfite solid or 1 mL of a 25% NaHSO3 solution was added, and the mixture was allowed to stand for 5 rain. The amount of bisulfite must be sufficient to destroy all the nitrite in the sample, which can be determined by applying the KI-Starch Test. The analysis should proceed as usual past this point. It is important to avoid Ion 371.00amu. from T0011301013.d Ion 377.00amu. from T0011301013.d Abundance Abundance acidifyingthe urine before adding the sodium bisulfite. 6OOOO 60000 It appears that the 40000 40000 degradation of THCCOOH occurs during the 20000 20000 acidification step of the 0 extraction procedure in the 13.8 14.0 14.2 14.4 13.8 14.0 14.2 14.4 absence of bisulfite. Time (min) Time (min) Figure 1 shows the ion chromatograms of a urine Ion 473.00amu. from T0011301013.d Ion 476.00amu. from T0011301013.d Abundance specimen containing 50 Abundance 24000 [~1 ng/mL THCCOOHand 16000/~l spiked with 2.5 mg/mL of 12000' Klear followedby GC-MS 8000 analysis according to the procedure published by 4000 / "~. E1Sohly et al. (2) using the 0 r--T-,~ - r--I --r~ i --~--r--r-'l--;~--r'-r-l--4'='P~ TMS derivative9The 13.8 14.0 14.2 14.4 13.8 14.0 14.2 14.4 "iTme(min) Time (rain) chromatograms show that neither the drug nor the Ion 488.00amu. from T0011301013.d internal standard could be Ion 494.00amu. from T0011301013.d Abundance Abundance identified because of the 16000 1 / 1 extremely low recovery. On 1 ooo the other hand, Figure 2 12000 t shows the same sample spiked in the same manner 40o0 ~ but with the addition of 250 0-1' ' ' I ' '-rl ' ' J I-' ' w--I--I-1--'~-mg of sodium bisulfite 5 13.8 14.0 14.2 14.4 Time(min) Time (rain) rain before extraction. Both the drug and the internal standard were recovered Figure 2. GC-MS analysisof a urine specimen containing 50 n~mL THCCOOH spiked with 2.5 m~rnL Klear and confirmation was but with the addition of 250 mg of sodium bisulfite 5 min before extraction. Ions monitored were as follows: m/z achieved without a 371,473, and 488 for THCCOOH and 377, 476, and 494 for internal standard. problem. i

i

180001 12~176176 t 84:!L

j

i

i

i

J/

0

16oooi

f\

400O1

/ \

--~

~,

t

'l-W~--"

I

,

,

,

r--z--r'-r'l--=,','

~--

241

Journal of Analytical Toxicology, Vol. 21, May/June 1997

Work is in progress to identify the degradation products of the potassium nitrite reaction with THCCOOH and to study the effect of long-term storage of urine specimens containing THCCOOH in the presence of Klear at different pH values.

M.A. E1Sohly,1,2S. Feng1, W.J. Kopycki1, T.P.Murphy1, A.B. Jones2, A. Davis3, and D. Carr3 1EISohlyLaboratories, Inc., Oxford, Mississippi; 2National Center for the Development of Natural Products and Department of Pharmaceutics, School of Pharmacy, University of Mississippi; and 3LabOne, Lenexa, Kansas

References 1. 2.

242

S.D. Brunk. False negative GC/MS assay for carboxy THC due to ibuprofen interference. J. Anal. Toxicol. 12:290-91 (1988). M.A. EISohly, T.L. Little, Jr., D.F. Stanford. Hexadeutero-11-nor-A9-tetrahydrocannabinol-9-carboxylic acid: A superior internal standard for the GC-MS analysis of Ag-THC acid metabolite in biological fluids. J. Anal. Toxicol. 16:188-91 (1992).