Ilpo Rasanen

Screening for basic drugs in hair of drug addicts by liquid chromatography/time-of-flight mass spectrometry.

Hair analysis in forensic and clinical toxicology has been strongly focused on drugs of abuse, and comprehensive, drug class-independent screening methods based on mass spectrometric detection have not been applied to date. In this study, a qualitative drug screening method by liquid chromatography coupled to time-of-flight mass spectrometry, earlier developed and evaluated for forensic toxicological urine analysis, was adapted for screening of basic drugs in hair. The method included alkaline hydrolysis, purification with mixed-mode solid phase extraction, and analysis by liquid chromatography coupled to time-of-flight mass spectrometry with automated data analysis and reporting. Identification was based on accurate mass, isotopic pattern fit, and retention time, if available. Analysis of 32 hair samples from deceased drug addicts revealed 35 different drugs. The drug classes identified included antidepressants, antipsychotics, antiepileptics, amphetamines, opioids, beta-blockers, a benzodiazepine, a hypnotic, a local anesthetic, an antiemetic, and an antipyretic analgesic. The findings were in good agreement with the findings in blood and urine by other methods. Moreover, information about previous drug use not evident in the analysis of other matrices was obtained in the majority (72%) of the cases. Tramadol was an especially predominant finding, suggesting tramadol abuse as an opioid substitute. One apparent false-positive finding was identified. The mean and median mass accuracies of positive findings were 2.3 and 1.8 ppm, corresponding to 0.5 and 0.4 mDa, respectively. Cutoff values for tramadol and methamphetamine in hair were 100 and 200 pg/mg, respectively. The method proved to be a simple and straightforward tool for comprehensive screening of basic drugs in hair.

Urine analysis of 3,4-methylenedioxypyrovalerone in opioid-dependent patients by gas chromatography-mass spectrometry.

A gas chromatography-mass spectrometry (GCMS) procedure was developed for the quantitative analysis of the new designer drug methylenedioxypyrovalerone (MDPV) in urine together with the common stimulants amphetamine, methamphetamine, and methylenedioxymethamphetamine (MDMA). The procedure involved electron ionization (EI) GCMS in the selected ion monitoring (SIM) mode after liquid-liquid extraction with toluene and derivatization with heptafluorobutyric acid anhydride. All MDPV findings were confirmed by positive chemical ionization GCMS in SIM mode. Positive chemical ionization-GCMS allowed the protonated molecule M+H+ m/z 276 to be used as a target ion with 3 abundant fragments as qualifier ions. By electron ionization-GCMS, the limit of quantification (LOQ) for MDPV was 0.02 mg/L; and for amphetamine, methamphetamine, and MDMA, the LOQ was 0.05 mg/L. The method was applied to monitoring urine samples from opioid-dependent patients undergoing opioid substitution treatment. Nine of the 34 urine samples (26%) analyzed were MDPV positive by the GCMS procedure. The positive samples were obtained from 2 female and 7 male patients with a mean age of 31 years. The median (range) MDPV concentration was 0.16 mg/L (0.04-3.9 mg/L) based on the 7 samples for which a numeric value was obtained, whereas the concentration was below the LOQ but above the limit of detection in 2 samples. The method revealed amphetamine in approximately 40% of the cases, and there was no statistical difference between the MDPV-positive and MDPV-negative groups. Urine amphetamine concentrations were on average 10 times higher than those of MDPV. The opioid-dependent patients used MDPV mainly as a substitute for amphetamine, judging from the laboratory findings of this study and the information from our patients.

Pharmacokinetics of Oxycodone After Intravenous, Buccal, Intramuscular and Gastric Administration in Children

ObjectiveTo evaluate the pharmacokinetics of four administration routes of oxycodone parenteral liquid (10 mg/mL), single intravenous and intramuscular injections and buccal and gastric administration, in children.Patients and participantsForty generally healthy children, aged 6–93 months, undergoing inpatient surgery.MethodsAfter induction of anaesthesia, children received a single dose of oxycodone 0.1 mg/kg intravenously (n = 9), intramuscularly (n = 10), buccally (n = 11) or via an orogastric tube into the stomach (n = 10). Regular blood samples were collected up to 12 hours, and plasma was analysed for oxycodone using gas chromatography-mass spectrometry (limit of quantification 1 µg/L).ResultsThe peak drug concentration observed was 57–110 (mean 82) µg/L after intravenous administration, 23–54 (34) µg/L after intramuscular administration, 3.9–14 (9.8) µg/L after buccal administration and 1.7–15 (9.2) µg/L after gastric administration. The time to peak concentration was 2–30 (16) minutes in the intramuscular group, 30–480 (221) minutes in the buccal group and 60–360 (193) minutes in the gastric group. The terminal elimination half-lives were closely similar in the four groups: 124–208 (163) minutes in the intravenous group, 162–227 (150) minutes in the intramuscular group, 73–234 (150) minutes in the buccal group and 80–246 (147) minutes in the gastric group. Area under the concentration-time curve (AUC) was 5037–8954 (6612) µg · min/L in the intravenous group, 3084–5524 (4473) µg · min/L in the intramuscular group, 1444-5560 (3658) µg · min/L in the buccal group and 692–3843 (2436) µg · min/L in the gastric group. The estimated bioavailability (AUC/mean intravenous AUC) of intramuscular oxycodone was 0.47–0.84 (0.68), that of buccal oxycodone 0.22–0.84 (0.55) and that of gastric oxycodone 0.10–0.58 (0.37).ConclusionThe pharmacokinetics of intravenous oxycodone in children aged 6–93 months are fairly similar to those reported in adults. Intramuscular administration provides relatively constant drug absorption, but after buccal and gastric administration the interindividual variation in the rate and extent of absorption is large.

Precise gas chromatography with retention time locking in comprehensive toxicological screening for drugs in blood

The long-term precision of three retention parameters, the absolute retention time (RT), the relative retention time related to dibenzepin (RRT), and the internal retention index based on the alkylfluoroaniline series (RI), were studied with 14 basic drugs on HP-5 and DB-17 columns with and without the use of the retention time locking option (RTL). Using the constant flow mode in all experiments, the RTL method was found to produce superior precision with all three retention parameters compared to the non-RTL method on each column. The results showed that RTL offers a significant advantage within a single instrument method, not only between methods, with CV<0.1% by RRT. Consequently, a dual-column gas chromatographic procedure with nitrogen-phosphorus detection was described for comprehensive screening for basic drugs in 1-ml whole blood samples. The method consisted of one-step liquid-liquid extraction with butyl acetate, identification using RRT in the RTL mode, and quantification based on single point calibration. The method allowed reliable screening and quantification of 124 basic drugs at therapeutic and toxic concentration levels in autopsy blood.

Performance of immunoassays in screening for opiates, cannabinoids and amphetamines in post-mortem blood

Several immunoassay methods for screening of abused drugs in whole blood were evaluated in post-mortem forensic toxicology. Blood samples known to be positive or negative for opiates, cannabinoids or amphetamines by gas chromatography-mass spectrometry (GC-MS) were analysed by EMIT II Plus and EMIT d.a.u., Syva RapidTest and Triage 8 after acetone precipitation. In these experiments, the EMIT immunoassay method was modified by using the Dade Behring VIVA analyser to detect substances more sensitively. Low concentrations of abused drugs were detected in blood samples. The sensitivities of the modified EMIT method for opiates, cannabinoids and amphetamines were 100, 86 and 98%, respectively, whereas the values were below 86% with the other methods. The specificities of all immunoassay methods for opiates and cannabinoids were 83% or above but 51-85% for amphetamines. Sample rejection occurred in a few cases with the EMIT amphetamine assays. The modified EMIT immunoassay system presented here seems to be useful for screening of drugs of abuse in post-mortem blood samples, especially when urine is not available.

Comparison of Oxycodone Pharmacokinetics after Buccal and Sublingual Administration in Children

ObjectiveWe evaluated and compared the pharmacokinetics of two oral administration routes of oxycodone parenteral liquid (10 mg/mL) - single buccal and sublingual administration - in 30 generally healthy awake children, aged 6–91 months.MethodsTwo groups of children undergoing inpatient surgery were enrolled. In a randomised fashion, children received a single dose of oxycodone 0.2 mg/kg buccally (n = 15) or sublingually (n = 15). Regular blood samples were collected for up to 12 hours, and plasma was analysed for oxycodone, oxymorphone and noroxycodone using gas chromatography-mass spectrometry.ResultsBioavailability was similar after administration at the two instillation sites. The area under the plasma concentration-time curve from time zero extrapolated to infinity (AUC∞) was 2400–8000 ng · min/mL (median 4200 ng · min/mL) in the buccal group and 2700-7900 ng · min/mL (median 5500 ng · min/mL) in the sublingual group. After buccal administration, maximum plasma concentration (Cmax) was 5.4–39 ng/mL (16 ng/mL) after buccal and 5.5–42 ng/mL (22 ng/mL) after sublingual administration. Twelve of the 15 children in both groups reached the oxycodone analgesic concentration of 12 ng/mL, which was sustained for 43–209 minutes (median 160 minutes) in the children with buccal oxycodone and for 32–262 minutes (median 175 minutes) in the children with sublingual oxycodone. The terminal elimination half-lives were closely similar in the two groups: 104–251 minutes (median 140 minutes) in the buccal group and 110–190 minutes (150 minutes) in the sublingual group.ConclusionThe results of this study show that in young children the absorption of oxycodone is similar after buccal and sublingual instillation.

Benzodiazepine findings in blood and urine by gas chromatography and immunoassay

Gas chromatography (GC) and immunoassay techniques applied to blood and urine specimens were compared for the screening of benzodiazepines in postmortem forensic toxicology. Five hundred and six such successive postmortem cases in which both urine and peripheral blood was sent for toxicological analysis by the medical examiners were selected. The urine specimens were tested by the Emit((R)) d.a.u. Benzodiazepine Assay, and in parallel, the blood and urine specimens were screened for benzodiazepine drugs and their metabolites by an established automated dual-column GC method. The lowest number of positives (153) was obtained when immunoassay was performed without enzyme hydrolysis. When urine samples were hydrolysed before immunoassay, the number of positives increased to 175. The highest number of positives (200) was obtained in urine by GC, and the screening of blood by GC yielded 185 quantitative results. Despite the urine GC screening produced the most positives, the quantitative screening of the blood by GC appears to be the most efficient approach in postmortem forensic toxicology, considering the fact that although urine findings confirm the presence of the drug, quantitative results in urine are irrelevant to acute toxicity.

Blood Levels of 3-methylfentanyl in 3 Fatal Poisoning Cases

Three fatal poisoning cases due to 3-methylfentanyl are described. In each case, the death was accidental and occurred after injection of the opioid combined with amphetamine, heroin, or other drugs. The victims’ ages, ranging from 30 to 41 years, were higher than those typically found in heroin poisonings in Finland. The blood concentrations of cis-3-methylfentanyl, measured here for the first time by a specific tandem mass spectrometric method, ranged from 0.3 to 0.9 μg/L (mean 0.5 μg/L). These values are significantly lower than the levels reported for α-methylfentanyl and fentanyl in fatal poisonings. Repeated seizures of fentanyl and its analogs have been reported in Europe close to the Russian border.

Analgesic Concentrations of Oxycodone – A Prospective Clinical PK/PD Study in Patients with Laparoscopic Cholecystectomy

The analgesic concentrations of oxycodone in acute post-operative pain management have not been established. Here, we have evaluated the minimum effective concentration (MEC) and the minimum effective analgesic concentration (MEAC) of oxycodone in pain after laparoscopic cholecystectomy (LCC) in 23 adult patients. The patients were provided with 0.1 mg/kg of oxycodone i.v. 10-15 min. before the end of surgery. After surgery, when the wound pain at rest was ≥3/10 and/or ≥5/10 during wound compression, a first blood sample was obtained (MEC). A second blood sample was obtained after titration with 2 mg i.v. of oxycodone to wound pain <3/10 at rest and <5/10 during wound compression (MEAC). A third blood sample was obtained at the recurrence of the wound pain (the second MEC), and the final blood sample when pain relief was obtained a second time (the second MEAC). At the first onset of pain (MEC), mean P-oxycodone was 21 ng/mL (95% CI 13-29 ng/mL). At the first pain relief (MEAC), P-oxycodone was 55 ng/mL (19-91 ng/mL). The second MEC was 34 ng/mL (11-57 ng/mL), and the second MEAC was 47 ng/mL (14-80 ng/mL). In conclusion, the estimated MEC, 20-35 ng/mL, and MEAC, 45-50 ng/mL, values of P-oxycodone in patients after LLC were significantly higher than those proposed previously. Early pain after LCC appeared to be a feasible method to estimate the analgesic efficacy of oxycodone in acute pain management.

Elevated formic acid concentrations in putrefied post-mortem blood and urine samples.

Formic acid (FA) concentration was measured in post-mortem blood and urine samples as methyl formate using a headspace in-tube extraction gas-chromatography-mass-spectrometry method. A total of 113 cases were analyzed, each including a blood and urine sample fortified with 1% sodium fluoride. The cases were divided into three groups: regular (n=59), putrefied (n=30), and methanol-positive (n=22) cases. There was no evidence of ante-mortem methanol consumption in the regular and putrefied cases. In regular cases, the mean (and median) FA concentrations were 0.04 g/l (0.04 g/l) and 0.06 g/l (0.04 g/l) in blood and urine, respectively. In putrefied cases, the mean (and median) FA concentrations were substantially higher, 0.24 g/l (0.22 g/l) and 0.25 g/l (0.15 g/l) in blood and urine, respectively. In three putrefied cases, FA concentration in blood exceeded 0.5 g/l, a level associated with fatal methanol poisoning. Ten putrefied cases were reanalyzed after 3-4 months storage, and no significant changes in FA concentrations were seen. These observations suggest that FA was formed by putrefaction during the post-mortem period, not during sample storage when sodium fluoride was added as a preservative. In methanol-positive cases, the mean (and median) FA concentrations were 0.80 g/l (0.88 g/l) and 3.4 g/l (3.3 g/l) in blood and urine, respectively, and the concentrations ranged from 0.19 to 1.0 g/l in blood and from 1.7 to 5.6 g/l in urine. The mean (and median) methanol concentrations in methanol-positive cases were 3.0 g/l (3.0 g/l) and 4.4 g/l (4.7 g/l) in blood and in urine, respectively. The highest methanol concentrations were 6.0 g/l and 8.7 g/l in blood and urine, respectively. No ethyl alcohol was found in the methanol-positive blood samples. Poor correlation was shown between blood and urine concentrations of FA. Poor correlations were also shown, in both blood and urine, between methanol and FA concentrations.