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Validation of an enzyme-linked immunosorbent assay screening method and a liquid chromatography-tandem mass spectrometry confirmation method for the identification and quantification of ketamine and norketamine in urine samples from Malaysia.
J Anal Toxicol. 2009 Jul-Aug; 33(6):310-21.JA

Abstract

An ELISA and a liquid chromatography-tandem mass spectrometry (LC-MS-MS) confirmation method were developed and validated for the identification and quantitation of ketamine and its major metabolite norketamine in urine samples. The Neogen ketamine microplate ELISA was optimized with respect to sample and enzyme conjugate volumes and the sample preincubation time before addition of the enzyme conjugate. The ELISA kit was validated to include an assessment of the dose-response curve, intra- and interday precision, limit of detection (LOD), and cross-reactivity. The sensitivity and specificity were calculated by comparison to the results from the validated LC-MS-MS confirmation method. An LC-MS-MS method was developed and validated with respect to LOD, lower limit of quantitation (LLOQ), linearity, recovery, intra- and interday precision, and matrix effects. The ELISA dose-response curve was a typical S-shaped binding curve, with a linear portion of the graph observed between 25 and 500 ng/mL for ketamine. The cross-reactivity of 200 ng/mL norketamine to ketamine was 2.1%, and no cross-reactivity was detected with 13 common drugs tested at 10,000 ng/mL. The ELISA LOD was calculated to be 5 ng/mL. Both intra- (n = 10) and interday (n = 50) precisions were below 5.0% at 25 ng/mL. The LOD for ketamine and norketamine was calculated statistically to be 0.6 ng/mL. The LLOQ values were also calculated statistically and were 1.9 ng/mL and 2.1 ng/mL for ketamine and norketamine, respectively. The test linearity was 0-1200 ng/mL with correlation coefficient (R(2)) > 0.99 for both analytes. Recoveries at 50, 500, and 1000 ng/mL range from 97.9% to 113.3%. Intra- (n = 5) and interday (n = 25) precisions between extracts for ketamine and norketamine were excellent (< 10%). Matrix effects analysis showed an average ion suppression of 5.7% for ketamine and an average ion enhancement of 13.0% for norketamine for urine samples collected from six individuals. A comparison of ELISA and LC-MS-MS results demonstrated a sensitivity, specificity, and efficiency of 100%. These results indicated that a cutoff value of 25 ng/mL ketamine in the ELISA screen is particularly suitable and reliable for urine testing in a forensic toxicology setting. Furthermore, both ketamine and norketamine were detected in all 34 urine samples collected from individuals socializing in pubs by the Royal Malaysian Police. Ketamine concentrations detected by LC-MS-MS ranged from 22 to 31,670 ng/mL, and norketamine concentrations ranged from 25 to 10,990 ng/mL. The concentrations of ketamine and norketamine detected in the samples are most ikely indicative of ketamine abuse.

Authors+Show Affiliations

Forensic Medicine and Science, Division of Cancer Sciences and Molecular Pathology, Faculty of Medicine, University of Glasgow, Scotland, G12 8QQ United Kingdom.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Validation Study

Language

eng

PubMed ID

19653934

Citation

Harun, Norlida, et al. "Validation of an Enzyme-linked Immunosorbent Assay Screening Method and a Liquid Chromatography-tandem Mass Spectrometry Confirmation Method for the Identification and Quantification of Ketamine and Norketamine in Urine Samples From Malaysia." Journal of Analytical Toxicology, vol. 33, no. 6, 2009, pp. 310-21.
Harun N, Anderson RA, Miller EI. Validation of an enzyme-linked immunosorbent assay screening method and a liquid chromatography-tandem mass spectrometry confirmation method for the identification and quantification of ketamine and norketamine in urine samples from Malaysia. J Anal Toxicol. 2009;33(6):310-21.
Harun, N., Anderson, R. A., & Miller, E. I. (2009). Validation of an enzyme-linked immunosorbent assay screening method and a liquid chromatography-tandem mass spectrometry confirmation method for the identification and quantification of ketamine and norketamine in urine samples from Malaysia. Journal of Analytical Toxicology, 33(6), 310-21.
Harun N, Anderson RA, Miller EI. Validation of an Enzyme-linked Immunosorbent Assay Screening Method and a Liquid Chromatography-tandem Mass Spectrometry Confirmation Method for the Identification and Quantification of Ketamine and Norketamine in Urine Samples From Malaysia. J Anal Toxicol. 2009 Jul-Aug;33(6):310-21. PubMed PMID: 19653934.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Validation of an enzyme-linked immunosorbent assay screening method and a liquid chromatography-tandem mass spectrometry confirmation method for the identification and quantification of ketamine and norketamine in urine samples from Malaysia. AU - Harun,Norlida, AU - Anderson,Robert A, AU - Miller,Eleanor I, PY - 2009/8/6/entrez PY - 2009/8/6/pubmed PY - 2009/9/15/medline SP - 310 EP - 21 JF - Journal of analytical toxicology JO - J Anal Toxicol VL - 33 IS - 6 N2 - An ELISA and a liquid chromatography-tandem mass spectrometry (LC-MS-MS) confirmation method were developed and validated for the identification and quantitation of ketamine and its major metabolite norketamine in urine samples. The Neogen ketamine microplate ELISA was optimized with respect to sample and enzyme conjugate volumes and the sample preincubation time before addition of the enzyme conjugate. The ELISA kit was validated to include an assessment of the dose-response curve, intra- and interday precision, limit of detection (LOD), and cross-reactivity. The sensitivity and specificity were calculated by comparison to the results from the validated LC-MS-MS confirmation method. An LC-MS-MS method was developed and validated with respect to LOD, lower limit of quantitation (LLOQ), linearity, recovery, intra- and interday precision, and matrix effects. The ELISA dose-response curve was a typical S-shaped binding curve, with a linear portion of the graph observed between 25 and 500 ng/mL for ketamine. The cross-reactivity of 200 ng/mL norketamine to ketamine was 2.1%, and no cross-reactivity was detected with 13 common drugs tested at 10,000 ng/mL. The ELISA LOD was calculated to be 5 ng/mL. Both intra- (n = 10) and interday (n = 50) precisions were below 5.0% at 25 ng/mL. The LOD for ketamine and norketamine was calculated statistically to be 0.6 ng/mL. The LLOQ values were also calculated statistically and were 1.9 ng/mL and 2.1 ng/mL for ketamine and norketamine, respectively. The test linearity was 0-1200 ng/mL with correlation coefficient (R(2)) > 0.99 for both analytes. Recoveries at 50, 500, and 1000 ng/mL range from 97.9% to 113.3%. Intra- (n = 5) and interday (n = 25) precisions between extracts for ketamine and norketamine were excellent (< 10%). Matrix effects analysis showed an average ion suppression of 5.7% for ketamine and an average ion enhancement of 13.0% for norketamine for urine samples collected from six individuals. A comparison of ELISA and LC-MS-MS results demonstrated a sensitivity, specificity, and efficiency of 100%. These results indicated that a cutoff value of 25 ng/mL ketamine in the ELISA screen is particularly suitable and reliable for urine testing in a forensic toxicology setting. Furthermore, both ketamine and norketamine were detected in all 34 urine samples collected from individuals socializing in pubs by the Royal Malaysian Police. Ketamine concentrations detected by LC-MS-MS ranged from 22 to 31,670 ng/mL, and norketamine concentrations ranged from 25 to 10,990 ng/mL. The concentrations of ketamine and norketamine detected in the samples are most ikely indicative of ketamine abuse. SN - 0146-4760 UR - https://www.unboundmedicine.com/medline/citation/19653934/Validation_of_an_enzyme_linked_immunosorbent_assay_screening_method_and_a_liquid_chromatography_tandem_mass_spectrometry_confirmation_method_for_the_identification_and_quantification_of_ketamine_and_norketamine_in_urine_samples_from_Malaysia_ L2 - https://academic.oup.com/jat/article-lookup/doi/10.1093/jat/33.6.310 DB - PRIME DP - Unbound Medicine ER -