Use of LC-HRMS in full scan-XIC mode for multi-analyte urine drug testing - a step towards a 'black-box' solution?

The influx of new psychoactive substances (NPS) has created a need for improved methods for drug testing in toxicology laboratories. The aim of this work was to design, validate and apply a multi-analyte liquid chromatography-high-resolution mass spectrometry (LC-HRMS) method for screening of 148 target analytes belonging to the NPS class, plant alkaloids and new psychoactive therapeutic drugs. The analytical method used a fivefold dilution of urine with nine deuterated internal standards and injection of 2 µl. The LC system involved a 2.0 µm 100 × 2.0 mm YMC-UltraHT Hydrosphere-C18 column and gradient elution with a flow rate of 0.5 ml/min and a total analysis time of 6.0 min. Solvent A consisted of 10 mmol/l ammonium formate and 0.005% formic acid, pH 4.8, and Solvent B was methanol with 10 mmol/l ammonium formate and 0.005% formic acid. The HRMS (Q Exactive, Thermo Scientific) used a heated electrospray interface and was operated in positive mode with 70 000 resolution. The scan range was 100-650 Da, and data for extracted ion chromatograms used ± 10 ppm tolerance. Product ion monitoring was applied for confirmation analysis and for some selected analytes also for screening. Method validation demonstrated limited influence from urine matrix, linear response within the measuring range (typically 0.1-1.0 µg/ml) and acceptable imprecision in quantification (CV <15%). A few analytes were found to be unstable in urine upon storage. The method was successfully applied for routine drug testing of 17 936 unknown samples, of which 2715 (15%) contained 52 of the 148 analytes. It is concluded that the method design based on simple dilution of urine and using LC-HRMS in extracted ion chromatogram mode may offer an analytical system for urine drug testing that fulfils the requirement of a 'black box' solution and can replace immunochemical screening applied on autoanalyzers. Copyright © 2017 John Wiley & Sons, Ltd.

Plasma concentration of ketorolac after local infiltration analgesia in hip arthroplasty.

BACKGROUND: Local infiltration analgesia (LIA) with local anaesthetic (ropivacaine), a nonsteroidal anti-inflammatory drug (ketorolac) and epinephrine after lower extremity arthroplasty has gained increasing popularity during the last decade. This method has certain advantages, which include minimal systemic side effects, faster post-operative mobilization, earlier post-operative discharge from hospital and less opioid consumption. However, information regarding plasma concentrations of ketorolac after LIA mixture is insufficient to predict the risk of renal impairment in patients subjected to arthroplasty. AIM: To determine the maximal plasma concentration and the exposure of ketorolac during the first 30 h following LIA in hip arthroplasty. METHODS: Thirteen patients scheduled for primary total hip arthroplasty with LIA (ropivacaine 200 mg, ketorolac 30 mg and epinephrine 0.5 mg in a volume of 106 ml) were included. Plasma concentration of ketorolac was quantified by liquid chromatography-mass spectrometry. In addition, we assessed the effect of increasing age and decreasing glomerular filtration rate on the maximal plasma concentration and the total exposure to ketorolac during 30 h. RESULTS: The range of the maximal plasma concentration, 0.3-2.2 mg/l, was detected 30 min-4 h after completing the infiltration. Similar plasma levels have been reported after intramuscular injection of the same dose of ketorolac to healthy elderly volunteers. CONCLUSION: Exposure to ketorolac after LIA may be comparable to an intramuscular injection of the same dose. Decision of dose reduction should be based on clinical assessment of risk factors.

Pharmacokinetics after a single intravenous dose of the opioid ketobemidone in neonates.

BACKGROUND: Ketobemidone is often used as an alternative to morphine in children in the Scandinavian countries. In an earlier study, we have examined the pharmacokinetic properties in children in different age groups but have not focused on neonates. The aim of this clinical trial was to explore the pharmacokinetics of ketobemidone in neonates. METHODS: Fifteen full-term neonates (eight females) from 37 gestational weeks at birth and scheduled for elective surgery were included in the trial. Their median age was 3 days (range 1-18 days). Ketobemidone hydrochloride was administered as a single intravenous bolus dose, and ketobemidone concentrations were measured by liquid chromatography-mass spectrometry over 10 h. Pharmacokinetic parameters were calculated with standard compartmental methods. RESULTS: The median (range) values for ketobemidone clearance, apparent volume of distribution, volume of central compartment, distribution half-life and elimination half-life were 0.46 (0.23-0.84) l/h/kg, 4.64 (3.50-7.31) l/kg, 1.71 (0.16-3.47) l/kg, 2.85 (1.04-10.78) min and 7.26 (3.5-11.3) h. CONCLUSION: Compared with our previous study in children older than 1 year of age, the elimination of ketobemidone appeared to be slower in full-term neonates. Despite a low pharmacokinetic variability of ketobemidone as observed in the present neonatal patient population, we recommend individualizing the dose of ketobemidone based on observations of analgesic efficacy.

Pharmacokinetics after an intravenous single dose of the opioid ketobemidone in children.

BACKGROUND: Ketobemidone is often used as an alternative to morphine in children in the Scandinavian countries. The aim of this clinical trial was to explore the pharmacokinetics of ketobemidone in children because these properties have not been reported previously. METHODS: Thirty children, newborn to 10 years, scheduled for elective surgery were included in the trial. Ketobemidone hydrochloride was administered as a single intravenous bolus dose and ketobemidone and norketobemidone concentrations were measured by LC-MS over 8 h. Pharmacokinetic parameters were determined using compartmental methods. RESULTS: Six children were excluded from pharmacokinetic analysis because of incomplete blood sampling. The values of ketobemidone clearance (l/h/kg) given as median (range) were 0.84 (0.29-3.0) in Group A (0-90 days), 0.89 (0.55-1.35) in Group B (1-2.5 years) and 0.74 (0.50-0.99) in Group C (7-10 years). The corresponding values for apparent volume of distribution (l/kg) were 4.4 (3.7-6.9) (Group A), 2.6 (2.0-5.6) (Group B) and 3.9 (2.7-5.0 (Group C), and for elimination half-life (h) 3.0 (1.4-8.9) (Group A), 2.0 (1.2-4.7) (Group B) and 3.7 (2.4-6.9) (Group C), respectively. In the two neonates the elimination half-life was almost 9 h. The metabolite norketobemidone did not reach levels above the limit of quantification (0.07 ng/ml) in any of the patients. CONCLUSION: The pharmacokinetic parameters of ketobemidone in children older than 1 month appear to be similar to those in adults. Because of the large interindividual variability of the pharmacokinetics in neonates, further studies especially in this age group are warranted.

Direct injection LC-MS/MS method for identification and quantification of amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine and 3,4-methylenedioxymethamphetamine in urine drug testing.

A method based on direct injection of diluted urine for the identification and quantification of amphetamine, methamphetamine, 3,4-methylenedioxymetamphetamine and 3,4-methylenedioxyamphetamine in human urine by electrospray ionisation liquid chromatography-tandem mass spectrometry was validated for use as a confirmation procedure in urine drug testing. Two deuterium labelled analogues, amphetamine-D5 and 3,4-methylenedioxymetamphetamine-D5, were used as internal standards. Twenty microliter aliquots of urine were mixed with 80 microL internal standard solution in autosampler vials and 10 microL was injected. The chromatographic system consisted of a 2.0 mmx100 mm C18 column and the gradient elution buffers used acetonitrile and 25 mmol/L formic acid. Two product ions produced from the protonated molecules were monitored in the selected reaction monitoring mode. The intra- and inter-assay variability (coefficient of variation) was between 5 and 16% for all analytes at 200 and 6000 ng/mL levels. Ion suppression occurred early after injection but did not affect the identification and quantification of the analytes in authentic urine samples. The method was further validated by comparison with a reference gas chromatographic-mass spectrometric method using 479 authentic urine samples. The two methods agreed almost completely (99.8%) regarding identified analytes when applying a 150 ng/mL reporting limit. Four deviating results were observed for 3,4-methylenedioxymethamphetamine and this was due to uncertainty in quantification around the reporting limit. For the quantitative results the slope of the regression lines were between 0.9769 and 1.0146, with correlation coefficients>0.9339. We conclude that the presented liquid chromatographic-tandem mass spectrometric method is robust and reliable, and suitable for use as a confirmation method in urine drug testing for amphetamines.

Validation of direct injection electrospray LC-MS/MS for confirmation of opiates in urine drug testing.

A method based on the direct injection of diluted urine for the identification and quantification of morphine, morphine-3-glucuronide, morphine-6-glucuronide, codeine, codeine-6-glucuronide, ethylmorphine, ethylmorphine-6-glucuronide and 6-acetylmorphine (6AM) in human urine by electrospray ionisation liquid chromatography-tandem mass spectrometry was validated for use as a confirmation procedure in urine drug testing. Four deuterium labelled analogues were used as internal standards: morphine-3-glucuronide-D3, morphine-D3, codeine-D3 and 6AM-D3. Twenty microlitre aliquots of urine were mixed with 80 mul of the internal standard solution in autosampler vials and 10 mul was injected. The chromatographic system consisted of a 2.0 x 100 mm C18 column and the gradient elution buffers used acetonitrile and 25 mmol/l formic acid. Two product ions produced from the protonated molecular ions were monitored in the selected reaction monitoring mode. The intra- and inter-assay variability (coefficient of variation) was below 10% at higher levels for all analytes, but at the reporting limits the variation was above 20% for 6AM, morphine-3-glucuronide and codeine-6-glucuronide. Ion suppression occurred early after injection but did not affect the identification and quantification of the analytes in authentic samples. The method was further validated by comparison with a reference gas chromatographic-mass spectrometric method using authentic urine samples. The two methods agreed almost completely (99%) regarding the identified analytes, but for the quantitative results there were slightly lower levels when measuring glucuronides directly as compared to total determination after hydrolysis by gas chromatography-mass spectrometry. We conclude that the presented liquid chromatographic-tandem mass spectrometric method is robust and reliable, and suitable for use as a confirmation method in urine drug testing for opiates

Determination of perhexiline and hydroxyperhexiline in plasma by liquid chromatography-mass spectrometry.

A method for the quantitative determination of perhexiline and its main hydroxylated metabolites in human plasma, based on liquid chromatography-mass spectrometry (LC-MS), was developed. The method used protein precipitation with acetonitrile followed by dilution with water and subsequent direct injection of the extract into the LC-MS system. Hexadiline was used as internal standard and the intra-assay coefficients of variation were