Method validation and application of a liquid chromatography-tandem mass spectrometry method for drugs of abuse testing in exhaled breath.

A mass spectrometric method for drugs of abuse testing in exhaled breath employing a sampling device collecting aerosol particles was developed and applied in routine use. Analytes covered were amphetamine, methamphetamine, 6-acetylmorphine, morphine, cocaine, benzoylecgonine, diazepam, oxazepam and tetrahydrocannabinol. The method involved eluting drugs from the collection filter with methanol, quantification using deuterated analogs as internal standards, reversed phase chromatography with gradient elution, positive electrospray ionization and monitoring of two product ions per analyte in selected reaction monitoring mode. The measuring range was 6.0-1000pg/filter. The intra- and inter-assay imprecision expressed as the coefficient of variation was less than 7%. Influence from matrix was noted for most compounds but was compensated for the use of co-eluting internal standards. The LLOQ was 6.0pg/filter with intra-assay CV <5% and accuracy within 99-102% for all analytes. No chromatographic interference was observed in 20 negative control samples. The LC-MS/MS method was successfully applied for measuring drugs in unknown samples collected for the purpose of drug testing. Among the 1096 analyzed samples analytical findings were made in breath in 39 cases (3.6%). Most frequently found substances were the following: amphetamine (25 cases) methamphetamine (10 cases), THC (8 cases), cocaine (4 cases), benzoylecgonine (2 cases) and diazepam (2 cases). In conclusion, a fully validated and robust screening method suitable for the routine measurement of drugs of abuse in exhaled breath with a simple procedure for specimen collection and sample preparation was successfully developed.

Determination of amphetamine and methylphenidate in exhaled breath of patients undergoing attention-deficit/hyperactivity disorder treatment.

OBJECTIVES: It has been discovered recently that exogenous substances are detectable in exhaled breath after intake. Exhaled breath therefore constitutes a new possible matrix in clinical pharmacology and toxicology. The present work was aimed at exploring this possibility further by a study on patients treated for attention-deficit/hyperactivity disorder with D-amphetamine and methylphenidate. METHODS: Thirteen patients (age range: 32-61 years; 5 women) were included in the study, and breath and urine samples were collected at different times in the dose interval. Analyses of breath and urine samples were done with liquid chromatography-mass spectrometry methods. Urine was examined for amphetamine, methylphenidate, and its metabolite ritalinic acid. RESULTS: Among the 9 patients who received D-amphetamine medication in daily doses of 20-100 mg, amphetamine was detected in all subjects in amounts ranging from 1200 to 30,800 picogram per filter. Among 8 patients receiving methylphenidate medication in daily doses of 80-400 mg, it was detected and quantified in 7 of the cases in amounts ranging from 150 to 10,400 picogram per filter and ritalinic acid was detected and quantified in 3 of the cases ranging from 35 to 360 picogram per filter. In 1 case, methylphenidate was only detectable in breath and urine, whereas ritalinic acid was quantifiable in urine, which could indicate noncompliance, with the 4 hours of dose regimen prescribed. In a number of cases, the sampling was performed 24 hours after the last dose intake. Identification of amphetamine and methylphenidate was based on correct chromatographic retention time and correct product ion ratio with detection performed in selected reaction monitoring mode. CONCLUSIONS: The results confirm that amphetamine is present in exhaled breath after intake and demonstrate for the first time the presence of methylphenidate and ritalinic acid after its intake. This gives further support to the potential use of exhaled breath for detecting drug intake.

Multicomponent LC-MS/MS screening method for detection of new psychoactive drugs, legal highs, in urine-experience from the Swedish population.

The advent of new not yet legally regulated psychoactive substances sold over the Internet has created a challenge for clinical toxicology and drug testing laboratories. The routine use of immunoassay screening may no longer be the optimal solution in many instances since the number of analytes covered is becoming insufficient. The aim of this work was to design, validate and apply a multi-component LC-MS/MS method suitable for screening of a large number of target analytes belonging to the class of new psychoactive substances - legal highs. The analytical method was using a five-fold dilution of urine with internal standard (pethidine-d5) and injection of 2µL. The chromatographic system was using a 1.7-µm 100mm×2.1mm Ethylene Bridged Hybrid (BEH) C18 column and gradient elution with a flow rate of 600µL/min. Solvent A consisted of 0.1% formic acid and Solvent B was 100% acetonitrile. The gradient elution application was designed to have a wide polarity coverage with total run time of 4.0min. The tandem mass spectrometer was using an electrospray interface and operated in positive mode. Selected reaction monitoring of two ion transitions was used for each of 26 analytes. Method validation demonstrated limited influence from urine matrix, linear response within the measuring range (0.1-10µg/mL), acceptable imprecision in quantification (CV<15%). Some analytes were found not to be stable in urine upon storage. The method was successfully applied in routine drug testing. A total of 87 positive samples with 100 analytical findings were found to contain O-desmethyl-cis-tramadol (mostly without mitragynine), methylenedioxypyrovalerone, 4-fluoroamphetamine, methoxetamine, desoxypipradol, 4-fluoromethcathinone, 5,6-methylenedioxy-2-aminoindane, 4-methylmethcathinone, 3-fluoromethcathinone, 4-hydroxy-N-methyl-N-ethyltryptamine, α-methylamino-butyrophenone and 4-methoxymethcathinone.

Detection of drugs of abuse in exhaled breath using a device for rapid collection: comparison with plasma, urine and self-reporting in 47 drug users.

Exhaled breath has recently been identified as a matrix for the detection of drugs of abuse. This work aims to further document this application using a new and simple collection device in patients following recovery from acute intoxication. Breath, plasma and urine samples were collected from 47 patients (38 males, age range 25-74) together with interview data. Analysis of breath and plasma samples was done by liquid chromatography-mass spectrometry methods. Urine was screened using immunochemical reagents and positive findings confirmed with liquid chromatography-mass spectrometry methods. The 12 analytes investigated were: methadone, amphetamine, methamphetamine, 6-acetylmorphine, morphine, benzoylecgonine, cocaine, diazepam, oxazepam, alprazolam, buprenorphine and tetrahydrocannabinol. In all 47 cases, recent intake of an abused substance prior to admission was reported, but in one case the substance (ketobemidone) was not investigated. In 40 of the remaining cases (87%) breath analysis gave a positive finding of any of the substances that were part of the analytical investigation. Identifications were based on correct chromatographic retention time and product ion ratios obtained in selected reaction monitoring mode. In general, data from breath, plasma, urine and self-reporting were in good agreement, but in 23% of the cases substances were detected that had not been self-reported. All substances covered were detected in a number of breath samples. Considering that breath sampling was often done about 24 h after intake, the detection rate was considered to be high for most substances. Analytes with low detection rates were benzodiazepines, and a further increase in analytical sensitivity is needed to overcome this. This study further supports use of exhaled breath as a new matrix in clinical toxicology.

Evaluation of a direct high-capacity target screening approach for urine drug testing using liquid chromatography-time-of-flight mass spectrometry.

In this study a rapid liquid chromatography-time-of-flight mass spectrometry method was developed, validated and applied in order to evaluate the potential of this technique for routine urine drug testing. Approximately 800 authentic patient samples were analyzed for amphetamines (amphetamine and methamphetamine), opiates (morphine, morphine-3-glucuronide, morphine-6-glucuronide, codeine and codeine-6-glucuronide) and buprenorphines (buprenorphine and buprenorphine-glucuronide) using immunochemical screening assays and mass spectrometry confirmation methods for comparison. The chromatographic application utilized a rapid gradient with high flow and a reversed phase column with 1.8 µm particles. Total analysis time was 4 min. The mass spectrometer operated with an electrospray interface in positive mode with a resolution power of >10,000 at m/z 956. The applied reporting limits were 100 ng/mL for amphetamines and opiates, and 5 ng/mL for buprenorphines, with lower limits of quantification were 2.8-41 ng/mL. Calibration curves showed a linear response with coefficients of correlation of 0.97-0.99. The intra- and interday imprecision in quantification at the reporting limits were <10% for all analytes but for buprenorphines <20%. Method validation data met performance criteria for a qualitative and quantitative method. The liquid chromatography-time-of-flight mass spectrometry method was found to be more selective than the immunochemical method by producing lower rates of false positives (0% for amphetamines and opiates; 3.2% for buprenorphines) and negatives (1.8% for amphetamines; 0.6% for opiates; 0% for buprenorphines). The overall agreement between the two screening methods was between 94.2 and 97.4%. Comparison of data with the confirmation (LC-MS) results for all individual 9 analytes showed that most deviating results were produced in samples with low levels of analytes. False negatives were mainly related to failure of detected peak to meet mass accuracy criteria (±20 mDa). False positives was related to presence of interfering peaks meeting mass accuracy and retention time criteria and occurred mainly at low levels. It is concluded that liquid chromatography-time-of-flight mass spectrometry has potential both as a complement and as replacement of immunochemical screening assays.

Detection of drugs of abuse in exhaled breath from users following recovery from intoxication.

It has recently been demonstrated that amphetamine, methadone and tetrahydrocannabinol are detectable in exhaled breath following intake. Exhaled breath, therefore, constitutes a new possible matrix for drugs-of-abuse testing. The present work aims to further explore this possibility by a study on patients treated for acute intoxication with abused drugs. Fifty-nine patients (44 males, age range 24-74) were included in the study, and breath, plasma and urine samples were collected following recovery, together with interview data. Analyses of breath and plasma samples were conducted with liquid chromatography-mass spectrometry methods. Urine was screened using immunochemical reagents and positive findings confirmed with liquid chromatography-mass spectrometry methods. The following analytes were investigated: methadone, amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine, codeine, 6-acetylmorphine, diazepam, oxazepam, morphine, benzoylecgonine, cocaine, buprenorphine and tetrahydrocannabinol. In 53 of the studied cases, recent intake of an abused substance prior to admission was reported. In 35 of these (66%), the breath analysis gave a positive finding. Identifications were based on correct chromatographic retention time and product ion ratios obtained in selected reaction monitoring mode. Generally, data from breath, plasma, urine and self-report were in agreement. Detected substances in breath included amphetamine, methamphetamine, buprenorphine, 6-acetylmorphine, morphine, codeine, methadone, tetrahydrocannabinol, diazepam, oxazepam and cocaine. Problem analytes with low detection rates were benzodiazepines and tetrahydrocannabinol. This study gives further support to the possibility of developing exhaled breath into a new matrix for drugs-of-abuse testing by extending the number of analytes that are documented to be detectable in breath.

A randomized study comparing plasma concentration of ropivacaine after local infiltration analgesia and femoral block in primary total knee arthroplasty.

Pain after total knee arthroplasty (TKA) is difficult to control. A recently developed and increasingly popular method for postoperative analgesia following knee and hip arthroplasty is Local Infiltration Analgesia (LIA) with ropivacaine, ketorolac and epinephrine. This method is considered to have certain advantages, which include administration at the site of traumatized tissue, minimal systemic side effects, faster postoperative mobilization, earlier postoperative discharge from hospital and less opioid consumption. One limitation, which may prevent the widespread use of LIA is the lack of information regarding plasma concentrations of ropivacaine and ketorolac. The aim of this academically initiated study was to detect any toxic or near-toxic plasma concentrations of ropivacaine and ketorolac following LIA after TKA. Methods Forty patients scheduled for primary total knee arthroplasty under spinal anaesthesia, were randomized to receive either local infiltration analgesia with a mixture of ropivacaine 300 mg, ketorolac 30mg and epinephrine or repeated femoral nerve block with ropivacaine in combination with three doses of 10mg intravenous ketorolac according to clinical routine. Plasma concentration of ropivacaine and ketorolac were quantified by liquid chromatography-mass spectrometry (LC-MS). Results The maximal detected ropivacaine plasma level in the LIA group was not statistically higher than in the femoral block group using the Mann-Whitney U-test (p = 0.08). However, the median concentration in the LIA group was significantly higher than in the femoral block group (p < 0.0001; Mann-Whitney U-test). The maximal plasma concentrations of ketorolac following administration of 30mg according to the LIA protocol were detected 1 h or 2 h after release of the tourniquet in the LIA group: 152-958 ng/ml (95% CI: 303-512 ng/ml; n = 20). The range of the plasma concentration of ketorolac 2-3 h after injection of a single dose of 10mg was 57-1216 ng/ml (95% CI: 162-420 ng/ml; n = 20). Conclusion During the first 24 h plasma concentration of ropivacaine seems to be lower after repeated femoral block than after LIA. Since the maximal ropivacaine level following LIA is detected around 4-6 h after release of the tourniquet, cardiac monitoring should cover this interval. Regarding ketorolac, our preliminary data indicate that the risk for concentration dependent side effects may be highest during the first hours after release of the tourniquet. Implication Femoral block may be the preferred method for postoperative analgesia in patients with increased risk for cardiac side effects from ropivacaine. Administration of a booster dose of ketorolac shortly after termination of the surgical procedure if LIA was used may result in an increased risk for toxicity.