Rapid Extraction and Qualitative Screening of 30 Drugs in Oral Fluid at Concentrations Recommended for the Investigation of DUID Cases.

A rapid, simple extraction method followed by qualitative screening using liquid chromatography-tandem mass spectrometry (LC-MS-MS) for drugs in oral fluid is presented. The decision points were selected to be at, or lower, than those recommended as Tier I compounds by the National Safety Council's Alcohol, Drugs and Impairment Division for toxicological investigation of driving under the influence of drug (DUID) cases and were also at, or lower, than those recommended by Substance Abuse and Mental Health Service Administration and the Department of Transportation for Federal workplace drug testing programs. The method included 30 drugs: delta-9-tetrahydrocannabinol, amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxyamphetamine, cocaine, benzoylecgonine, carisoprodol, meprobamate, zolpidem, alprazolam, clonazepam, 7-aminoclonazepam, diazepam, nordiazepam, lorazepam, oxazepam, temazepam, codeine, morphine, 6-acetylmorphine, buprenorphine, fentanyl, hydrocodone, hydromorphone, oxycodone, oxymorphone, methadone, tramadol and phencyclidine. Phencyclidine was included because it is in the Federal workplace program even though it is considered a Tier II drug for DUID cases. A liquid-liquid extraction method using isopropanol, hexane and ethyl acetate to extract drugs from the oral fluid-buffer mix collected in a Quantisal™ device, followed by LC-MS-MS screening, was developed and validated according to ANSI/ASB 2019 Standard Practices for Method Validation in Forensic Toxicology. Interference studies, limit of detection, precision at the decision point, ionization suppression/enhancement and processed sample stability were determined for each drug. The method was successfully applied to proficiency specimens and routine samples received in the laboratory.

Cannabinoids in Oral Fluid: Limiting Potential Sources of Cannabidiol Conversion to Δ9- and Δ8-Tetrahydrocannabinol.

In late 2019, the National Laboratory Certification Program (NLCP) published an article reporting on the potential analytical conversion of 7-carboxy cannabidiol (CBD-COOH) to 11-nor-9-carboxy-Δ9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) in urine samples. The same conversion is possible in oral fluid with the parent analyte cannabidiol (CBD) converting to Δ9-tetrahydrocannabinol (Δ9-THC) and Δ8-tetrahydrocannabinol (Δ8-THC) under strong acidic conditions. With the recent rise in states legalizing the use of THC and the availability of products containing only CBD, unless the analytical in vitro conversions are controlled, the detection of Δ9-THC or Δ8-THC in oral fluid may not clarify whether the donor was using a CBD product or licit or illicit THC product. Authentic oral fluid samples submitted for cannabinoid analysis were subjected to multiple sample preparation procedures and extraction methods to determine the conditions that allow CBD to convert to THC. CBD single analyte controls prepared from a certified THC-free source were added to the batch to monitor the rate of conversion. Samples were prepared using a base hydrolysis, solid phase extraction, derivatization and analysis by liquid chromatography with tandem mass spectrometry (LC-MS-MS). The base hydrolysis and derivatization were tested independently and did not contribute to the conversion rate. Adjusting the pH of the sample preparation and extraction from pH 2.0 to pH 5.0 changed the conversion rate from 5 to 1%. A pH of 6.0 was not strong enough to extract the cannabinoids efficiently. Removing the acid component of the preparation and extraction procedure eliminated the conversion to THC; however, this did reduce the analyte recovery depending on which extraction column was used. Processing time also contributed to the conversion rate. With smaller trial runs, conversion was not always seen, but with larger validation batches low-level conversion of 1-2% was observed. A fully validated LC-MS-MS method utilizing solid-phase extraction was developed for CBD, Δ9-THC, Δ8-THC and cannabinol. The method specifically targets those analytes found in oral fluid after CBD administration and those that are seen during in vitro CBD conversion. CBD administration was performed using a certified THC-free CBD control.

Analysis of Drugs in Oral Fluid Using LC-MS/MS.

Oral fluid analysis for drugs is increasingly used in a variety of testing areas: pain management and medication monitoring, parole and probation situations, driving under the influence of drugs (DUID), therapeutic drug monitoring, and testing for drugs in the workplace. The sample collection itself is straightforward, rapid, observable, and noninvasive, requiring no special facilities (compared to urine) or medical personnel (compared to blood). The pH of saliva is slightly acidic relative to blood; therefore, drugs which are more basic tend to be present in higher concentration in oral fluid than in blood: cocaine, amphetamines, oxycodone, tramadol, buprenorphine, methadone, and fentanyl. Conversely, acidic drugs and drugs which are strongly protein bound have lower concentrations in oral fluid than in blood: examples include benzodiazepines, barbiturates, and carisoprodol. Because of the low volume of specimen available for analysis and the drug concentrations present (generally much lower than those in urine), efficient extraction methods and sensitive confirmation procedures are necessary for routine analysis of drugs in oral fluid. In this chapter, solid-phase extraction methods are described for a variety of drugs with liquid chromatography-tandem mass spectrometry detection.

Roadside drug testing: An evaluation of the Alere DDS® 2 mobile test system.

The number of drivers using drugs has increased over the last few years, and is likely to continue its upward trend. Testing drivers for alcohol use is routine and standardized, but the same is not true for the identification of driving under the influence of drugs (DUID). The Drug Evaluation and Classification Program (DECP) was developed to train police officers to recognize the signs and symptoms of recent drug use and remains an invaluable program; however, there are insufficient numbers of these highly trained drug recognition experts (DREs) available to attend every potential drug involved traffic incident. While blood and urine samples are used to test for drugs in a driver, both have disadvantages, particularly as they pertain to the length of time required after a traffic stop to sample collection. Therefore, the development of oral fluid testing devices which can be operated at the roadside and have the potential to assist officers in the identification of drug use is a major advancement in DUID cases. This project evaluated the performance of one instrumental oral fluid roadside testing device (Alere DDS®2) compared to DRE opinion, oral fluid laboratory-based analysis, and routine blood testing. The results showed that there was a good correlation with DRE observations and the device performance was >80% in all drug categories compared to laboratory-based analytical testing, both in oral fluid and blood, with few exceptions. The instrument can be considered a useful tool to assist law enforcement in identifying a drugged driver. Because the device does not test for all potentially impairing drugs, the opinion of the police officer regarding the condition of the driver should still be considered the most important aspect for arrest and further action.

Validation of a New Recombinant Antibody Fragment (rFab)-Based Homogeneous Enzyme Immunoassay for the Highly Specific Detection of 6-Acetylmorphine in Urine.

The detection of 6-acetylmorphine (6-AM) in urine by immunoassay methods is challenging due to its short half-life and its similarity in structure to many commonly abused opiates that are often present at very high concentrations in urine. Current 6-AM homogeneous enzyme immunoassays use lyophilized reagents because of the instability of 6-AM in water or lack of the required specificity due to high cross-reactivity with morphine. A new 6-AM rFab-based homogeneous enzyme immunoassay (HEIA) has been developed with highly improved specificity. Using a cutoff concentration of 10 ng/mL, morphine or morphine glucuronides did not produce a positive signal up to 300,000 or 1,000,000 ng/mL, respectively. Assay imprecision (n = 80) was less than 1.5% using four replicates per day for 20 days over the range 0-20 ng/mL. Cross-reactivity with structurally related or non-related compounds was assessed at concentrations up to 1,000,000 ng/mL. Interferences from endogenous compounds at ±25% cutoff were also performed at the concentrations ranging from 100,000 to 500,000 ng/mL. The effect of varied pH values on assay performance at ±25% cutoff was investigated; no false-positive or false-negative results were observed between pH 4 and -11. Based on the analysis of 149 authentic urine samples, the accuracy of the 6-AM HEIA compared with LC-MS-MS was 100%. These results demonstrated that rFab can be suitable for traditional HEIA with desired detection sensitivity and stability.

Analysis of tetrahydrocannabinol and its metabolite, 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid, in oral fluid using liquid chromatography with tandem mass spectrometry.

This paper describes the determination of tetrahydrocannabinol (THC) and its metabolite, 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) in oral fluid using solid-phase extraction and liquid chromatography with tandem mass spectral detection (LC-MS-MS) and its application to proficiency specimens. The method employs collection of oral fluid with the Quantisal™ device, base hydrolysis, solid-phase extraction and LC-MS-MS in positive ion electrospray mode. Because the concentration of the metabolite in oral fluid is quite low, extremely sensitive analytical methods are necessary. The requisite sensitivity was achieved by a simple, rapid derivatization of the compound after extraction. The derivatization conditions did not affect parent THC. The method was fully validated using standard parameters including linearity, sensitivity, accuracy, intra-day and inter-day imprecision, drug recovery from the collection pad, limit of quantitation, limit of detection and matrix effects. The procedure was applied to oral fluid proficiency specimens previously analyzed to assess the stability of THC-COOH.

Determination of carisoprodol and meprobamate in oral fluid.

The determination of carisoprodol and its metabolite meprobamate in oral fluid using solid-phase extraction and liquid chromatography with tandem mass spectral detection (LC-MS-MS) and its application to authentic specimens is described. The method employs collection of oral fluid with the Quantisal device, extraction using cation exchange/hydrophobic solid-phase columns, and LC-MS-MS in positive ion electrospray mode. The method was fully validated using various parameters, including selectivity, linearity, accuracy, intra-day and inter-day imprecision, drug recovery from the collection pad, limit of quantitation and matrix effects. The method was applied to both routine research specimens and an authentic specimen taken from an individual prescribed a daily dose of 350 mg carisoprodol following surgery.

Disposition of opioids in oral fluid: Importance of chromatography and mass spectral transitions in LC-MS/MS.

The use of prescription pain relievers, specifically opioids, has been increasing over the last few years. Oral fluid is easier to collect than urine, is difficult to adulterate, and is a reflection of free drug in the body, so its analysis is becoming more widespread in the monitoring of opioids. The demethylated metabolites of oxycodone, hydrocodone, and codeine are present at higher concentrations in oral fluid than oxymorphone, hydromorphone, and morphine, respectively; therefore, their detection in saliva indicates ingestion of the medication rather than diversion, and should be included in the analysis of opioids in this matrix. Since the compounds have the same nominal molecular weights, the same M + H(+) precursor ions in positive electrospray mode, and potentially identical collisionally activated fragmentation patterns, the importance of chromatography to separate the various opioids as well as the selection of mass spectral transitions is critical for correct identification. A procedure for the simultaneous determination of 12 opioid related compounds in oral fluid using liquid chromatography with tandem mass spectrometry (LC-MS/MS) is presented. The recovery of opioids from the collection device was over 80% at 20 ng/ml; intra-day imprecision was less than 6.8%; inter-day imprecision less than 6.2%. In authentic specimens, the predominant metabolite of oxycodone was noroxycodone; for specimens containing codeine, no morphine was detected; and for hydrocodone positives, norhydrocodone was detected at significantly higher levels than hydromorphone. The importance of monitoring specific mass spectral transitions and chromatographic separation is demonstrated.

Synthetic cannabinoids in oral fluid.

At the end of 2010, the U.S. Drug Enforcement Administration (DEA) used its emergency scheduling authority to temporarily control five chemicals, JWH-018, JWH-073, JWH-200, CP-47497, and cannabicyclohexanol (CP-47497 C8), often referred to as "Spice", K2, or "synthetic cannabinoids" because of their reported cannabis-like effects. JWH-250 is commonly encountered, and HU-210 was already controlled, so these were also included in the research. We report the first analytical procedure for the simultaneous determination of these compounds in oral fluid specimens collected with the Quantisal™ device using solid-phase extraction and liquid chromatography with tandem mass spectrometry. The method was validated and applied to specimens taken from two individuals who had purchased the synthetic compounds while still legally available in the U.S. After a single session of smoking "Blueberry Posh", the peak concentration of JWH-018 detected was 35 µg/L 20 min after smoking; JWH-018 was still detectable 12 h after a single intake. After a single session of smoking "Black Mamba", JWH-018 was detected with a peak concentration of 5 µg/L after 20 min. In this subject, the compound was not detectable after 12 h.

Selection of an immunoassay screening cutoff concentration for opioids in oral fluid.

A retrospective analysis of data from oral fluid specimens was conducted in order to identify a relevant cutoff concentration for opiates and/or synthetic opiates in oral fluid. Previously proposed regulations from the Substance Abuse and Mental Health Services Administration (SAMHSA) have recommended 40 µg/L as a cutoff concentration. In this study, data from oral fluid specimens collected using the Quantisal™ device and screened with enzyme linked immunosorbent assays (ELISA) for both opiates and oxycodone were retrospectively assessed for screen positives > 20 µg/L and those between negative and 20 µg/L. Specimens identified at these concentrations were then analyzed using liquid chromatography with tandem mass spectral detection using a fully validated procedure. Overall, 156 positive specimens were identified using 40 µg/L; 191 specimens using 20 µg/L; and 241 specimens between negative and 20 µg/L. Specifically, the number of 6-acetylmorphine (6-AM) positives increased from 10 to 16; morphine 4 to 9; codeine from 11 to 19; oxycodone from 56 to 74; hydrocodone from 73 to 119; and hydromorphone from 2 to 4 when specimens with enzyme inhibition between negative and 20 µg/L were analyzed. For workplace testing where only codeine, morphine, and 6-AM are considered, the use of a lowered cutoff concentration produced significant increases in the positive rate.

Cannabinoids in oral fluid following passive exposure to marijuana smoke.

The concentration of tetrahydrocannabinol (THC) and its main metabolite 11-nor-Δ(9)-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) as well as cannabinol (CBN), and cannabidiol (CBD) were measured in oral fluid following realistic exposure to marijuana in a Dutch coffee-shop. Ten healthy subjects, who were not marijuana smokers, volunteered to spend 3h in two different coffee shops in Groningen, The Netherlands. Subjects gave two oral fluid specimens at each time point: before entering the store, after 20 min, 40 min, 1h, 2h, and 3h of exposure. The specimens were collected outside the shop. Volunteers left the shop completely after 3h and also provided specimens approximately 12-22 h after beginning the exposure. The oral fluid specimens were subjected to immunoassay screening; confirmation for THC, cannabinol and cannabidiol using GC/MS; and THC-COOH using two-dimensional GC-GC/MS. THC was detectable in all oral fluid specimens taken 3h after exposure to smoke from recreationally used marijuana. In 50% of the volunteers, the concentration at the 3h time-point exceeded 4 ng/mL of THC, which is the current recommended cut-off concentration for immunoassay screening; the concentration of THC in 70% of the oral fluid specimens exceeded 2 ng/mL, currently proposed as the confirmatory cut-off concentration. THC-COOH was not detected in any specimens from passively exposed individuals. Therefore it is recommended that in order to avoid false positive oral fluid results assigned to marijuana use, by analyzing for only THC, the metabolite THC-COOH should also be monitored.

Validation of a new homogeneous immunoassay for the detection of carisoprodol in urine.

The objective of this project was to validate a new high-throughput homogeneous enzyme immunoassay (HEIA) for the rapid detection of carisoprodol in human urine. Carisoprodol (Soma(®)) and meprobamate are widely prescribed as musculoskeletal pain relief drugs and are listed as one of the 10 most frequently identified drugs associated with DUI cases. Carisoprodol has a short elimination half-life of 1-3 h; however, its major active metabolite, meprobamate, has a longer elimination half-life of 6-17 h. As a result, it is important for an immunoassay to cross-react with both compounds. The advantage of this new assay is that cutoff concentrations can be adjusted between 100 and 500 ng/mL. The reportable range was 25 to 1000 ng/mL for carisoprodol and 50 to 10,000 ng/mL for meprobamate. The intraday coefficient of variation (% CV) for the semi-quantitative assay was less than 1%. The homogeneous assay was validated with a total of 86 urine samples previously analyzed by liquid chromatography-tandem mass spectrometry with carisoprodol concentrations ranging from 50 to 10,000 ng/mL. The accuracy was found to be 100% when immunoassay cutoff concentrations of carisoprodol and meprobamate were set at 100 and 1000 ng/mL, respectively.

Development of a homogeneous immunoassay for the detection of fentanyl in urine.

OBJECTIVE: Fentanyl is an extremely potent synthetic opioid that is widely used for chronic pain treatment; it is highly addictive and prone to abuse. The objective is to develop a high throughput homogeneous enzyme immunoassay (HEIA) for the rapid detection of fentanyl in human urine. METHODS: The HEIA is based on an immunoassay format in which both the antibody and enzyme-drug conjugate are in ready-to-use solution. In the absence of the target analyte in the specimen, enzyme-labeled drug conjugate binds to the antibody and results in a decrease of the enzyme (G6PDH) activity; hence there is lower absorbance at 340 nm. If the target analyte is present in the specimen, it competes with the enzyme-labeled drug to bind to limited amount of specific antibody that result in more enzyme activity and yields an increased absorbance at 340 nm. A polyclonal "in-house" antibody was selected that is capable of measuring fentanyl at low concentrations thus the assay detection limit was determined to be 1 ng/mL. The assay was validated with clinical urine specimens that previously confirmed positively or negatively for fentanyl/norfentanyl by LC-MS/MS. RESULTS: The intra-day (n = 20) and inter-day (n = 100) precision of the assay was less than 1% CV. No interferences from structurally unrelated and commonly ingested drugs were observed at a concentration of 10,000 ng/mL. A total of 209 LC-MS/MS confirmed urine specimens (149 positive and 57 negative samples) were analyzed by HEIA. The sensitivity, specificity, and accuracy values were 99%, 95%, and 98% respectively. CONCLUSION: This paper describes the development of a highly sensitive homogenous enzyme immunoassay for detecting fentanyl in urine at a cut-off concentration of 2 ng/mL.

Antidepressant drugs in oral fluid using liquid chromatography-tandem mass spectrometry.

An analytical procedure for the determination of widely prescribed drugs for the treatment of depression and anxiety disorders, including amitriptyline, cyclobenzaprine, imipramine, dothiepin, doxepin, fluoxetine, sertraline, trimipramine, protriptyline, chlorpromazine, clomipramine, and some of their metabolites (nortriptyline, desmethyldoxepin, desipramine, desmethyltrimipramine, norclomipramine) in oral fluid has been developed and validated using liquid chromatography with tandem mass spectral detection. The oral fluid samples were collected using the Quantisal device and screened with enzyme-linked immunosorbent assay. Any drugs present were quantified using mixed-mode solid-phase extraction followed by mass spectrometric detection in positive electrospray ionization mode. For confirmation, two transitions were monitored, and the ratio between the two was required to be within 20% of the known calibration standard. Because of the worldwide shortage of acetonitrile, which was first reported in October 2008, the mobile phase was optimized to use methanol as the organic component. For all compounds, the lower limit of quantitation was 5 ng/mL; the intraday precision ranged from 2.9 to 8.2% (n = 6); interday precision from 1.5 to 6.2% (n = 30) at a concentration of 40 ng/mL. The percentage recovery of antidepressants from the oral fluid collection pad was calculated at a concentration of 40 ng/mL and ranged from 51.4 to 84.1% (n = 6). The aim of the study was to develop a confirmatory procedure for drugs in oral fluid that had been identified as presumptively positive for antidepressants and related compounds. The methods were applied to research oral fluid specimens received into our facility for testing.

Semi-quantitative analysis of drugs of abuse, including tetrahydrocannabinol in hair using aqueous extraction and immunoassay.

A semi-quantitative analytical screening procedure for the determination of cocaine, amphetamines, opiates, and delta-9-tetrahydrocannabinol in hair has been developed. The procedure employs an aqueous extraction buffer, uses only 10mg of hair, requires 2h of incubation for the extraction to occur, and multiple drug classes can be screened using enzyme linked immunosorbent assays. Hair calibration standards were prepared around the recommended cut-off concentrations of the Society of Hair Testing. All drug classes showed excellent linearity over the concentration range tested, indicating that immunochemical screening can be used in a semi-quantitative mode for hair analysis using an aqueous buffer, rapid extraction and a small amount of hair.

Determination of tapentadol and its metabolite N-desmethyltapentadol in urine and oral fluid using liquid chromatography with tandem mass spectral detection.

An analytical procedure for the determination of the new pain medication tapentadol and its main metabolite N-desmethyltapentadol (DMT), in urine and oral fluid has been developed and validated using liquid chromatography with tandem mass spectral detection (LC-MS-MS). Oral fluid was collected using Quantisal™ devices, and drugs present were quantified using solid-phase extraction followed by LC-MS-MS. For confirmation, two transitions were monitored and one ratio determined which had to be within 20% of that of the known calibration standard. For tapentadol, 222.1 > 107 was used as the quantifying transition; 222.1 > 121 for the qualifier. For DMT, 208.1 > 107 was used for quantification; 208.1 > 121 as the qualifier. For saliva, the linear range was 10-100 ng/mL; the lower limit of quantitation (LLOQ) was 10 ng/mL; the intraday precision was 3.6% (n = 6) and interday precision was 13.6% (n = 24). The recovery of tapentadol and DMT from the oral fluid collection pad was > 99%. For urine, the specimens were diluted and injected directly into the LC-MS-MS. The LLOQ was 50 ng/mL; the intraday and interday precisions were 2.1% and 4.4%, respectively, for tapentadol and 2.9% and 5.7%, respectively, for DMT. This is the first analytical procedure for tapentadol and DMT in urine and oral fluid.

Development of a homogeneous immunoassay for the detection of zolpidem in urine.

Sleep disorders are common conditions that affect about 40 million people in the U.S every year, the most common of which is insomnia, which is characterized by difficulty falling or staying asleep. Zolpidem (Ambien) is a non-benzodiazepine prescription drug that is used to treat insomnia and is often preferred over the commonly used benzodiazepines due to a lesser side effect profile. This is because the non-benzodiazepine binding is more selective to GABA-A receptors versus the non-selective binding of benzodiazepines. With the increasing popularity of non-benzodiazepines, drug abuse and driving-while-impaired cases involving sleep-inducing drugs have risen. Therefore, a highly sensitive and rapid homogeneous immunoassay (EMIT-type assay) has been developed for the detection of zolpidem in urine. The zolpidem antibody is highly specific and does not cross-react with other newer sleep aids such as zopiclone and zaleplon. This assay has a detection limit of 5 ng/mL for zolpidem in urine. Further evaluation of this assay using liquid chromatography-tandem mass spectrometry (LC-MS-MS) analysis of authentic urine samples demonstrated that the accuracy of the assay is greater than 90%. Because this assay is designed to measure the non-conjugated drug in urine, it resulted in simplification for gas chromatography-MS or LC-MS-MS confirmation methods that do not require urine hydrolysis before solid-phase extraction or liquid-liquid extraction.

Highly sensitive and specific liquid chromatography- tandem mass spectrometry method for testing ractopamine in cow and sheep urine.

Ractopamine belongs to a group of beta-agonist compounds. It is a forbidden food additive in most countries because of the many reported collective intoxication outbreaks in humans. Because the drug can be rapidly metabolized and eliminated from animal body, the objective of this study was to develop an efficient and sensitive liquid chromatography-tandem mass spectrometry (LC-MS-MS) analytical method for the detection of ractopamine with cutoff below 1 ng/mL to extend the detection window after withdrawal of the drug and at the same time to meet the requirements of zero-tolerance policy set by most countries. After solid-phase cleanup of the non-hydrolyzed and hydrolyzed urine samples, the residues were dissolved in methanol and analyzed directly by LC-MS-MS. Two transitions were monitored and one ratio determined. Samples reported as positive were required to have the ratio of the transitions within 20% of that determined from known calibration standards. The monitoring of the qualifying transition and requirement for its presence within a specific ratio to the primary ion has the potential of limiting the sensitivity of the assay. The limits of detection and quantitation of the assay were 0.1 and 0.25 ng/mL, respectively. The intraday precisions at 0.25 and 35 ng/mL were 4.88% and 0.95%, respectively. Interday precisions were 5.64% and 0.9% at the same concentrations. The percentage recoveries at 0.25 and 0.5 ng/mL were 117.8% and 108.4%, respectively.

Quantitation of tetrahydrocannabinol in hair using immunoassay and liquid chromatography with tandem mass spectrometric detection.

A quantitative analytical procedure for the determination of Delta(9)-tetrahydrocannabinol (THC) in hair has been developed and validated using liquid chromatography with tandem mass spectral detection (LC-MS/MS). Specimens that were determined as containing cannabinoids following immunoassay testing were quantified using solid-phase extraction followed by liquid chromatographic separation and tandem mass spectral detection in positive electrospray ionization mode. For confirmation, two transitions were monitored and one ratio determined. Samples being reported as positive were required to have both transitions present, the ratio of quantifying transition to qualifying transition being within 20% of that determined from known calibration standards. The limit of quantitation and the limit of detection was 10 pg/mg. The percentage recovery of the THC from hair at 20 pg/mg was 56% and a matrix effect of the hair showed an ion suppression percentage of -51%. The immunochemical screening method was performed following a rapid aqueous extraction, requiring only 10 mg of hair; the confirmatory procedure required 20 mg of hair. The methods were applied to proficiency specimens from the Society of Hair Testing, which had been received in August 2008.

Tetrahydrocannabinol and two of its metabolites in whole blood using liquid chromatography-tandem mass spectrometry.

An analytical procedure for the determination of Delta9-tetrahydrocannabinol (THC), 11-nor-9-carboxy-Delta9-tetrahydrocannabinol (THCA), and 11-hydroxy-Delta9-tetrahydrocannabinol (11-OH-THC) in whole blood has been developed and validated using liquid chromatography with tandem mass spectral detection (MS). Cannabinoids present in the blood samples were quantified using solid-phase extraction followed by MS detection in positive electrospray ionization mode. For confirmation, two transitions were monitored and one ratio determined. Samples being reported as positive were required to have both transitions present, the ratio of quantifying transition to qualifying transition being within 20% of that determined from known calibration standards. The monitoring of the qualifying transition and requirement for its presence within a specific ratio to the primary ion has the potential of limiting the sensitivity of the assay, however, the additional confidence in the final result as well as forensic defensibility were considered to be of greater importance. The limit of quantitation was 0.5 ng/mL for THC, 5 ng/mL for THCA, and 2 ng/mL for 11-OH-THC. The limit of detection was 0.5 ng/mL for THC, 4 ng/mL for THCA, and 1 ng/mL for 11-OH-THC. The percentage recovery of the cannabinoids from whole blood at a concentration of 5 ng/mL was 71.5% for THC, 64.5% for 11-OH-THC, and 61.2% for THCA (n = 3).