Quantitative analysis of drugs in hair by UHPLC high resolution mass spectrometry.

Liquid chromatographic methods coupled to high resolution mass spectrometry are increasingly used to identify compounds in various matrices including hair but there are few recommendations regarding the parameters and their criteria to identify a compound. In this study we present a method for the identification and quantification of a range of drugs and discuss the parameters used to identify a compound with high resolution mass spectrometry. Drugs were extracted from hair by incubation in a buffer:solvent mixture at 37°C during 18h. Analysis was performed on a chromatographic system comprised of an Agilent 6550 QTOF coupled to a 1290 Infinity UHPLC system. High resolution accurate mass data were acquired in the All Ions mode and exported into Mass Hunter Quantitative software for quantitation and identification using qualifier fragment ions. Validation included selectivity, matrix effects, calibration range, within day and between day precision and accuracy. The analytes were 7-amino-flunitrazepam, 7-amino-clonazepam, 7-amino-nitrazepam, acetylmorphine, alimemazine, alprazolam, amphetamine, benzoylecgonine, buprenorphine, diazepam, ethylmorphine, fentanyl, hydroxyzine, ketobemidone, codeine, cocaine, MDMA, methadone, methamphetamine, morphine, oxycodone, promethazine, propiomazine, propoxyphene, tramadol, zaleplone, zolpidem, and zopiclone. As proof of concept, hair from 29 authentic post mortem cases were analysed. The calibration range was established between 0.05ng/mg to 5.0ng/mg for all analytes except fentanyl (0.02-2.0), buprenorphine (0.04-2.0), and ketobemidone (0.05-4.0) as well as for alimemazine, amphetamine, cocaine, methadone, and promethazine (0.10-5.0). For all analytes, the accuracy of the fortified pooled hair matrix was 84-108% at the low level and 89-106% at the high level. The within series precisions were between 1.4 and 6.7% and the between series precisions were between 1.4 and 10.1%. From the 29 autopsy cases, 121 positive findings were encountered from 23 of the analytes in concentrations similar to those previously published. We conclude that the developed method proved precise and accurate and that it had sufficient performance for the purpose of detecting regular use of drugs or treatment with prescription drugs. To identify a compound we recommend the use of ion ratios as a complement to instrument software "matching scores".

A screening method for 30 drugs in hair using ultrahigh-performance liquid chromatography time-of-flight mass spectrometry.

OBJECTIVES: The objectives of this study were to develop and to validate a qualitative screening method that met the new Society of Hair Testing (SoHT) guideline criteria for thresholds. METHODS: Extraction of 20 mg hair was performed by a previously validated procedure using overnight incubation in a mixture of methanol:acetonitrile:formiate buffer pH 3 (10:10:80). Analysis was performed on an Agilent 6540 quadrupole time-of-flight mass spectrometer in combination with an Agilent 1290 Infinity ultrahigh-performance liquid chromatography system. Separation was achieved with a 12-minute linear gradient chromatography on a high-strength silica T3 column at acidic conditions. An in-house database containing 30 compounds from the groups amphetamines, opiates, opioids, cocaine, benzodiazepines, and other sedatives including 6 deuterated internal standards was built by analyzing solutions from certified standards. Data were extracted using mass accuracy of ± 10 ppm, retention time deviation of ± 0.15 minutes, and area of ≥ 30,000 counts. Identification was based on scoring of retention time, accurate mass measurement, and isotopic pattern. Validation included selectivity, repeatability of analyte area, and the scoring parameters at the proposed thresholds and a method comparison with the present liquid chromatography-mass spectrometry-mass spectrometry method using 50 authentic hair samples. A daily cutoff calibrator was used to identify positive samples. RESULTS: All cutoffs could be met with imprecisions of less than 5% for most parameters and analytes. Hair from drug-free subjects did not produce any positive results and the method comparison agreed in more than 90% of the cases. CONCLUSIONS: We conclude that the developed method meets the criteria of the new SoHT guidelines for screening cutoffs. Even though no thresholds have been suggested for benzodiazepines, we conclude that thresholds between 0.05 and 0.1 ng/mg should be sufficient to determine regular use of these substances.

Hair analysis for drugs in driver's license regranting. A Swedish pilot study.

When being convicted for petty drug offence or driving under the influence of drugs in Sweden, the driving license may be suspended. To regain the license, the person has to prove that he or she has been drug free during an observation period. This is controlled by urine samples taken at several occasions. However, the risk of manipulation and the risk of false negative urine samples are high. In addition, many people find it difficult or embarrassing to urinate when observed. Hair sampling might therefore be a welcome option to this procedure, with its easy sampling and minimal risk of manipulation. The longer detection window may also provide better information to the physician. The aim of this work was to evaluate if clients preferred hair samples to urine and to investigate practical and interpretive problems or advantages with hair samples. Ninety-nine hair samples and 198 urine samples were collected from 84 clients during the 12 month study period. Hair samples were divided into either one segment (0-3 cm) or two segments (0-3 and 3-6 cm) depending on the length. The hair samples were screened with LC-MS-MS for 20 drugs and confirmation of positive results were performed with GC-MS or LC-MS-MS. The results were compared to urine samples taken at two occasions during the observation period. To cover the timeframe of the urine samples hair was collected 2 weeks after the second sample. The urine samples were analysed with immunochemical screening and positive results confirmed with GC-MS or LC-MS-MS. Seventy-four clients presented with negative results in both urine and hair. Hair analysis identified illegal drugs at seven different occasions whereas urine failed to identify any illegal drugs. However the thresholds used may still be too high to find sporadic use as clients that admitted to use drugs sporadically presented with drug concentrations lower than the agreed thresholds but above the limit of detection. This implicates that the physician must have an understanding and knowledge of the limitations of the screening methods used. Another important outcome was that the clients approved of hair sampling considering it a better means to prove their drug abstinence. In addition, both the clients and the clinicians thought hair sampling easier than urine sampling. We believe that hair analysis can offer several advantages compared to urine analysis for clinicians working with driving license regranting.

Urinary detection times and excretion patterns of flunitrazepam and its metabolites after a single oral dose.

We investigated the excretion profiles of flunitrazepam metabolites in urine after a single dose. Sixteen volunteers received either 0.5 or 2.0 mg flunitrazepam. Urine samples were collected after 2, 4, 6, 8, 12, 24, 48, 72, 96, 120, 240, and 336 h. Samples were screened using CEDIA (300 microg/L cutoff) and quantitated using liquid chromatography-tandem mass spectrometry. The cutoff was 0.5 microg/L for flunitrazepam, N-desmethylflunitrazepam, 7-aminoflunitrazepam, 7-aminodesmethylflunitrazepam, 7-acetamidoflunitrazepam, and 7-acetamidodesmethylflunitrazepam. None of the subjects receiving 0.5 mg were screened positive, and only 23 of 102 samples from the subjects given 2.0 mg were positive with CEDIA. The predominant metabolites were 7-aminoflunitrazepam and 7-aminodesmethylflunitrazepam. For all subjects given the low dose, 7-aminoflunitrazepam was detected up to 120 h, and for two subjects for more than 240 h. Seven subjects given the high dose were positive up to 240 h for 7-aminoflunitrazepam. We conclude that the ratio 7-aminodesmethylflunitrazepam to 7-aminoflunitrazepam increased with time, independent of dose, and may be used to estimate the time of intake. For some low-dose subjects, the metabolite concentrations in the early samples were low and a chromatographic method may fail to detect the intake. We think laboratories should consider this when advising police and hospitals about sampling as well as when they set up strategies for analysis.