Stability of 21 Cocaine, Opioid and Benzodiazepine Drug Analytes in Spiked Meconium at Three Temperatures.

In this study, the stability of 21 cocaine, opioid and benzodiazepine analytes in spiked meconium was investigated at three storage temperatures: 4°C, room temperature (RT), and 37°C (body temperature). The drugs/metabolites included were hydrocodone, hydromorphone, codeine, morphine, 6-acetylmorphine (6-AM), oxycodone, oxymorphone, cocaine, cocaethylene, benzoylecgonine, m-hydroxybenzoylecgonine, diazepam, oxazepam, temazepam, nordiazepam, chlordiazepoxide, lorazepam, alprazolam, alpha-hydroxyalprazolam, clonazepam, 7-aminoclonazepam, midazolam, alpha-hydroxymidazolam and zolpidem. Drug testing was performed using mass spectrometry methods that were validated for clinical use. After 2 weeks of storage, a substantial loss was observed in the concentrations of 7-aminoclonazepam (48.4% at 4°C and 71.5% at RT), and chlordiazepoxide (59.5% at RT). A slight decrease was observed in the concentrations of alprazolam (20.9% at 4°C), clonazepam (24.5% at 4°C), chlordiazepoxide (23.5% at 4°C), midazolam (20.8% at 4°C), nordiazepam (22.8% at RT), and alpha-hydroxyalprazolam (20.7% at 4°C). At 37°C, the concentrations of chlordiazepoxide, 7-aminoclonazepam, lorazepam, oxazepam, nordiazepam and temazepam decreased by 81.4%, 86.8%, 56.5%, 59.9%, 45.4% and 31.7%, respectively, after 2 weeks. 6-AM was observed to be unstable regardless of storage temperatures. For morphine, a 33.3% increase at 4°C and a 23.4% increase at RT were observed after 2 weeks, respectively, possibly due to 6-AM degradation, while no changes ≥20% were observed at 37°C. All other analytes were stable up to 2 weeks at all three storage temperatures (concentration changes <20%). The stability of select drug analytes in authentic clinical meconium specimens was consistent with that observed in spiked meconium. In conclusion, some drugs in meconium may not be stable for long periods of time. Sample storage conditions are an important consideration in the context of detection windows and interpreting drug-testing results in meconium. To the best of our knowledge, this is the first stability study of cocaine, opioids and benzodiazepines in meconium concerning the effects of storage temperatures.

One Hundred False-Positive Amphetamine Specimens Characterized by Liquid Chromatography Time-of-Flight Mass Spectrometry.

Some amphetamine (AMP) and ecstacy (MDMA) urine immunoassay (IA) kits are prone to false-positive results due to poor specificity of the antibody. We employed two techniques, high-resolution mass spectrometry (HRMS) and an in silico structure search, to identify compounds likely to cause false-positive results. Hundred false-positive IA specimens for AMP and/or MDMA were analyzed by an Agilent 6230 time-of-flight (TOF) mass spectrometer. Separately, SciFinder (Chemical Abstracts) was used as an in silico structure search to generate a library of compounds that are known to cross-react with AMP/MDMA IAs. Chemical formulas and exact masses of 145 structures were then compared against masses identified by TOF. Compounds known to have cross-reactivity with the IAs were identified in the structure-based search. The chemical formulas and exact masses of 145 structures (of 20 chemical formulas) were compared against masses identified by TOF. Urine analysis by HRMS correlates accurate mass with chemical formulae, but provides little information regarding compound structure. Structural data of targeted antigens can be utilized to correlate HRMS-derived chemical formulas with structural analogs.

Quantitation of Total Buprenorphine and Norbuprenorphine in Meconium by LC-MS/MS.

Buprenorphine (Suboxone, Zubsolv, Buprenex, Butrans, etc.) is an opioid drug that has been used to treat opioid dependence on an outpatient basis, and is also prescribed for managing moderate to severe pain. Pregnant women may be prescribed buprenorphine as part of a treatment plan for opioid addiction. This chapter quantitates buprenorphine and norbuprenorphine in meconium by liquid chromatography tandem mass spectrometry (LC-MS/MS).

Quantitation of Buprenorphine, Norbuprenorphine, Buprenorphine Glucuronide, Norbuprenorphine Glucuronide, and Naloxone in Urine by LC-MS/MS.

Buprenorphine is an opioid drug that has been used to treat opioid dependence on an outpatient basis, and is also prescribed for managing moderate to severe pain. Some formulations of buprenorphine also contain naloxone to discourage misuse. The major metabolite of buprenorphine is norbuprenorphine. Both compounds are pharmacologically active and both are extensively metabolized to their glucuronide conjugates, which are also active metabolites. Direct quantitation of the glucuronide conjugates in conjunction with free buprenorphine, norbuprenorphine, and naloxone in urine can distinguish compliance with prescribed therapy from specimen adulteration intended to mimic compliance with prescribed buprenorphine. This chapter quantitates buprenorphine, norbuprenorphine, their glucuronide conjugates and naloxone directly in urine by liquid chromatography tandem mass spectrometry (LC-MS/MS). Urine is pretreated with formic acid and undergoes solid phase extraction (SPE) prior to analysis by LC-MS/MS.

A hybrid approach to urine drug testing using high-resolution mass spectrometry and select immunoassays.

OBJECTIVES: The major objective of this research was to propose a simplified approach for the evaluation of medication adherence in chronic pain management patients, using liquid chromatography time-of-flight (TOF) mass spectrometry, performed in parallel with select homogeneous enzyme immunoassays (HEIAs). We called it a "hybrid" approach to urine drug testing. METHODS: The hybrid approach was defined based on anticipated positivity rates, availability of commercial reagents for HEIAs, and assay performance, particularly analytical sensitivity and specificity for drug(s) of interest. Subsequent to implementation of the hybrid approach, time to result was compared with that observed with other urine drug testing approaches. RESULTS: Opioids, benzodiazepines, zolpidem, amphetamine-like stimulants, and methylphenidate metabolite were detected by TOF mass spectrometry to maximize specificity and sensitivity of these 37 drug analytes. Barbiturates, cannabinoid metabolite, carisoprodol, cocaine metabolite, ethyl glucuronide, methadone, phencyclidine, propoxyphene, and tramadol were detected by HEIAs that performed adequately and/or for which positivity rates were very low. Time to result was significantly reduced compared with the traditional approach. CONCLUSIONS: The hybrid approach to urine drug testing provides a simplified and analytically specific testing process that minimizes the need for secondary confirmation.

Comparison of drug detection by three quadrupole time-of-flight mass spectrometry platforms.

Urine and plasma specimens fortified with 82 drugs and metabolites were prepared and analyzed by liquid chromatography quadrupole time-of-flight mass spectrometry (QTOF) instrumentation from three different vendors using the instrument manufacturers' methods and workflows for drug screening. No prior knowledge about the compounds included or their concentrations were provided. Samples were prepared and sent for analysis on a TripleTOF(®) 5600 system, a 6530 QTOF and a Xevo(®) G2-S QTof. All three platforms performed well with >90% of compounds detected in one set of spiked plasma samples, and 79-88% for a second set of spiked plasma and two sets of spiked urine samples.

Detection of neonatal drug exposure using umbilical cord tissue and liquid chromatography time-of-flight mass spectrometry.

BACKGROUND: A method for qualitative detection of 57 drugs and metabolites in umbilical cord tissue using liquid chromatography time-of-flight (TOF) mass spectrometry is described. METHODS: Results from 32 deidentified positive specimens analyzed by an outside laboratory using "screen with reflex to confirmation" testing were compared with TOF results. In addition, 57 umbilical cord tissue specimens paired with corresponding chart review data and 37 with meconium test results were analyzed by TOF. Urine drug test results from mother (n = 18) and neonate (n = 30) were included if available. Cutoff concentrations, recovery, and matrix effects were determined by analyzing fortified drug-free cord tissue and negative specimens. Cutoffs (in nanograms per gram) ranged from 1 to 10 for opioids and opioid antagonists, 5-10 for benzodiazepines and nonbenzodiazepine hypnotics, 20-40 for barbiturates, 8 for stimulants, and 4 for phencyclidine. Adequate sensitivity for the detection of cannabis exposure could not be realized with this method. CONCLUSIONS: Liquid chromatography time-of-flight mass spectrometry can provide accurate and sensitive detection of in utero drug exposure using umbilical cord tissue.

Demystifying analytical approaches for urine drug testing to evaluate medication adherence in chronic pain management.

This comprehensive review of analytical methods used for urine drug testing for the support of pain management describes the methods, their strengths and limitations, and types of analyses used in clinical laboratories today. Specific applications to analysis of opioid levels are addressed. Qualitative versus quantitative testing, immunoassays, chromatographic methods, and spectrometry are discussed. The importance of proper urine sample collection and processing is addressed. Analytical explanations for unexpected results are described. This article describes the scientific basis for urine drug testing providing information which will allow clinicians to differentiate between valid and questionable claims for urine drug testing to monitor medication adherence among chronic pain patients.

Rapid screening for 67 drugs and metabolites in serum or plasma by accurate-mass LC-TOF-MS.

Sixty-seven drugs and metabolites were detected in serum or plasma using a fast (7.5 min) liquid chromatography time-of-flight mass spectrometry (LC-TOF-MS) method. This method was developed as a blood drug screen, with emphasis on the detection of common drugs of abuse and drugs used to manage chronic pain. Qualitative drug detection may identify a drug exposure, assure patient adherence with prescribed therapy and document abstinence from non-prescribed medications. Compound identification is based on chromatographic retention time, mass, isotope spacing and isotope abundance. Data analysis software (Agilent) generates a compound score based on how well these observed criteria matched theoretical and empirical values. The method was validated using fortified samples and 299 residual patient specimens (920 positive results). All results were confirmed by gas chromatography-MS or LC-tandem MS. The accuracy of positive results (samples meeting all qualitative criteria for retention time, mass and compound score) was >90% for drugs and/or metabolites, except for two benzodiazepines. There were 35 false positive results (seven compounds, 3.8%) that could be distinguished by retention time and/or absence of metabolites. The most frequent was 6-acetylmorphine in the absence of morphine. The LC-TOF-MS targeted screening method presented represents a sensitive and specific technology for drug screening of serum or plasma.

Sensitive UPLC-MS-MS assay for 21 benzodiazepine drugs and metabolites, zolpidem and zopiclone in serum or plasma.

This paper reports an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) method to quantitate 21 benzodiazepines, zolpidem and zopiclone in serum and plasma. After liquid-liquid extraction, an Acquity UPLC with a TQ Detector and BEH C18 column was used (Waters, Milford, MA). The injection-to-injection run time was 7.5 min. Forty-eight authentic serum and plasma patient specimens were analyzed and results compared to those obtained using a previously published method. Average r(2) values for linearity (1 to 1,000 ng/mL over five days) were all above 0.995, except α-hydroxytriazolam (0.993). Intra-day and inter-day relative standard deviation values were within ± 15% and the percent deviation from the expected concentrations were within ± 11%. Recovery ranged from 62 to 89%. Matrix effects ranged from -28% to +6%. The limits of detection were 1 ng/mL, except for lorazepam, nordiazepam, oxazepam and temazepam (5 ng/mL). Ion ratios were ± 15% for all analytes. For authentic patient specimens (n = 48, 76 positive results), there was excellent correlation between the UPLC-MS-MS results and the previous method. The best least-squares fit had an equation of y = 1.0708x + 1.6521, r(2) = 0.9822. This UPLC-MS-MS method is suitable for the quantification of benzodiazepines and hypnotics in serum and plasma, and offers fast, reliable and sensitive results.

Patterns of free (unconjugated) buprenorphine, norbuprenorphine, and their glucuronides in urine using liquid chromatography-tandem mass spectrometry.

Patterns of buprenorphine and metabolites were examined in 1946 positive urine samples analyzed by liquid chromatography-tandem mass spectrometry for free (unconjugated) buprenorphine and norbuprenorphine (quantitative, 2 to 1000 ng/mL) and buprenorphine-glucuronide (B3G) and norbuprenorphine-glucuronide (N3G) (semi-quantitative, 5 to 1000 ng/mL). Two distribution patterns predominated with 49.1% positive for norbuprenorphine, B3G, and N3G and 41.6% positive for buprenorphine, norbuprenorphine, B3G, and N3G. Buprenorphine, positive in 45.5% of samples, was mostly < 5 ng/mL (median 6.1 ng/mL), but 9.8% were > 1000 ng/mL. Norbuprenorphine, B3G, and N3G had semi-Gaussian distributions with medians of 64.7, 108, and 432 ng/mL, respectively. With buprenorphine < 100 ng/mL (767 samples) or ≥ 100 ng/mL (19 quantifiable samples), the respective median metabolic ratios (free norbuprenorphine/free buprenorphine) were 25.0 and 0.15. In 12 retested "> 1000 ng/mL" buprenorphine samples, free buprenorphine was 4160 to 39,400 ng/mL and free naloxone 2140 to 9560 ng/mL. In 87 subsequent samples with buprenorphine < 20 ng/mL, naloxone concentrations were < 50 ng/mL. Concentrations of buprenorphine > 100 ng/mL (particularly with low metabolite concentrations) are suspect of urine adulteration with medication (4% in the database) that can be checked in most cases by concurrent analysis for naloxone.

Drugs of abuse detection in meconium: a comparison between ELISA and biochip microarray.

The results of meconium specimens and fortified samples screened for drugs of abuse by both enzyme-linked immunosorbent assay (ELISA, Immunalysis) and biochip microarray (Randox) methods were compared. The ELISA method was semi-automated using a TECAN Genesis. The Randox assay used the Randox Evidence Investigator system. Previously validated gas chromatography-mass spectrometry (GC-MS), GC-GC-MS, or liquid chromatography-MS-MS methods were used for confirmation and quantitation. Results from the two techniques compared well. Agreement of the Randox assay was greater than 90% when compared to the ELISA assay for all drug classes except cannabinoids (88%). Specificity of the biochip assay was slightly better for amphetamines and cocaine.

Nicotine and metabolites in paired umbilical cord tissue and meconium specimens.

Umbilical cord tissue was studied as a means of detecting prenatal exposure to nicotine. This was accomplished by comparing the presence and concentration of nicotine as well as nicotine metabolites in both umbilical cord tissue and paired meconium samples with maternal smoking histories obtained by self-report. Nicotine and metabolites (cotinine, 3-hydroxycotinine, nornicotine, and anabasine) were detected and quantitated using liquid chromatography-tandem mass spectroscopy. Between June and September 2009, 19 women with a tobacco exposure history (either first- or second-hand tobacco smoke exposure during pregnancy) were consented for the study. A questionnaire was completed to document nicotine exposure during each trimester of pregnancy. All infants were delivered at term (38 weeks or greater) and paired umbilical cord tissue (10-cm segment or greater) and meconium were obtained. Nicotine and 3-hydroxycotinine were most prominent in meconium, whereas cotinine and 3-hydroxycotinine were most prominent in the umbilical cord. Concentrations of all three analytes were generally higher in meconium. Nornicotine was detected only in meconium, at very low concentrations, and anabasine was not detected in either specimen. All analyte concentrations were lowest when the mother stated she quit smoking early in pregnancy or had only second-hand exposure, and detection was poor if exposure was limited to the first or second trimesters. Although different nicotine and metabolite patterns exist in meconium versus umbilical cord tissue, this work indicates that either specimen can be used to detect third-trimester fetal nicotine exposure.

LC-MS/MS analysis of 13 benzodiazepines and metabolites in urine, serum, plasma, and meconium.

We describe a single method for the detection and quantitation of 13 commonly prescribed benzodiazepines and metabolites: alpha-hydroxyalprazolam, alpha-hydroxyethylflurazepam, alpha-hydroxytriazolam, alprazolam, desalkylflurazepam, diazepam, lorazepam, midazolam, nordiazepam, oxazepam, temazepam, clonazepam and 7-aminoclonazepam in urine, serum, plasma, and meconium. The urine and meconium specimens undergo enzyme hydrolysis to convert the compounds of interest to their free form. All specimens are prepared for analysis using solid-phase extraction (SPE), analyzed using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), and quantified using a three-point calibration curve. Deuterated analogs of all 13 analytes are included as internal standards. The instrument is operated in multiple reaction-monitoring (MRM) mode with an electrospray ionization (ESI) source in positive ionization mode. Urine and meconium specimens have matrix-matched calibrators and controls. The serum and plasma specimens are quantified using the urine calibrators but employing plasma-based controls. Oxazepam glucuronide is used as a hydrolysis control.

Comparison of drugs of abuse detection in meconium by EMIT II and ELISA.

The results of meconium specimens and fortified samples screened for drugs of abuse by both enzyme multiplied immunoassay technique (EMIT((R) )II) and enzyme-linked immunosorbent assay (ELISA) methods were compared. The sample preparation for the ELISA screen was a simple buffer extraction versus a lengthy and more laborious sample preparation procedure for the EMIT II screen. The ELISA method was automated using a TECAN Genesis. The EMIT II analysis was automated with an Olympus AU400e. The opioid screen was calibrated with hydromorphone and the benzodiazepine screen was calibrated with clonazepam to maximize detection for these analytes. Previously validated gas chromatography-mass spectrometry (GC-MS), two-dimensional GC-MS, or liquid chromatography-tandem MS methods were used for confirmation. Results from the two techniques compared well. Agreement of the ELISA assay was greater than 90% when compared to EMIT II for all drug classes except barbiturates and benzodiazepines. ELISA appears to be more sensitive than EMIT II for the detection of amphetamines, methadone, propoxyphene, and cocaine. ELISA compared well to EMIT II for cannabinoids, opioids, and PCP. Specificity of the ELISA assay was slightly better for PCP and opioids. EMIT II appears to be more sensitive for the detection of barbiturates and benzodiazepines. The ELISA method reduced turnaround time by 50% compared to the EMIT II method.

Evaluation of a new ELISA kit for the detection of benzodiazepines in meconium.

Two versions of enzyme-linked immunosorbent assay (ELISA) kits designed for the detection of benzodiazepine drugs and metabolites (Immunalysis) were evaluated for use with meconium specimens. One was an older kit, and one was a new replacement kit developed for better detection of several commonly prescribed benzodiazepines and metabolites. The kits were evaluated by analyzing 68 patient specimens previously analyzed by liquid chromatography-tandem mass spectrometry and eight quality control samples. In addition to the recommended calibrator (oxazepam), clonazepam was evaluated as an alternate calibrator for the new kit. Detection and sensitivity for some analytes was improved using the new ELISA kit, but was reduced for others. The new kit using clonazepam as the calibrator provided the most sensitive assay for detection of the 11 benzodiazepines and metabolites reported here.

Quantitation of benzodiazepines in urine, serum, plasma, and meconium by LC-MS-MS.

A single method for confirmation and quantitation of a panel of commonly prescribed benzodiazepines and metabolites, alpha-hydroxyalprazolam, alpha-hydroxyethylflurazepam, alpha-hydroxytriazolam, alprazolam, desalkylflurazepam, diazepam, lorazepam, midazolam, nordiazepam, oxazepam, temazepam, clonazepam, and 7-aminoclonazepam, was developed for three specimen types, urine, serum/plasma, and meconium. Quantitation was by liquid chromatography tandem-mass spectrometry (LC-MS-MS) using a Waters Alliance-Quattro Micro system. The instrument was operated in multiple reaction monitoring mode with an electrospray ionization source in positive ionization mode. The method was evaluated for recovery, imprecision, linearity, analytical measurement range, specificity, and carryover. Average recovery and imprecision (within-run, between-run, and total % CV) were within +/- 15% of the target concentrations for urine (10 to 5000 ng/mL) and serum/plasma (10 to 2500 ng/mL) and within +/- 20% for meconium (10 to 5000 ng/g). In all, 205 patient specimens were analyzed, and the results compared to a previous in-house gas chromatography-MS method or LC-MS-MS results from an outside laboratory. Oxazepam glucuronide was evaluated as a hydrolysis control for the urine and meconium specimens.

Confirmation of cannabinoids in meconium using two-dimensional gas chromatography with mass spectrometry detection.

Meconium has become the specimen of choice for determining fetal exposure to drugs of abuse, but its physical complexity can cause interferences from matrix effects. A new method to determine 9-carboxy-11-nor-Delta(9)-THC (9-THCA) and 11-hydroxy-Delta(9)-THC (11-OH-THC) using two-dimensional (2D) GC-MS was developed to reduce interferences and carryover. The method was validated using 70 spiked samples prepared in drug-free meconium and 46 residual patient specimens that were confirmed to contain cannabinoids. Ten patient specimens that failed to confirm due to interferences using the previous GC-MS method were analyzed using the new 2D method and 9-THCA was quantitated in all ten samples. The 2D GC-MS method improved chromatography which significantly reduced interferences and carryover when compared to the previous GC-MS method.

Effects of elevated temperature and mobile phase composition on a novel C18 silica column.

A novel polydentate C18 silica column was evaluated at an elevated temperature under acidic, basic, and neutral mobile phase conditions using ACN and methanol as the mobile phase organic modifier. The temperature range was 40-200 degrees C. The mobile phase compositions were from 0 to 80% organic-aqueous v/v and the mobile phase pH levels were between 2 and 12. The maximum operating temperature of the column was affected by the amount and type of organic modifier used in the mobile phase. Under neutral conditions, the column showed good column thermal stability at temperatures ranging between 120 and 200 degrees C in methanol-water and ACN-water solvent systems. At pH 2 and 3, the column performed well up to about 160 degrees C at two fixed ACN-buffer compositions. Under basic conditions at elevated temperatures, the column material deteriorated more quickly, but still remained stable up to 100 degrees C at pH 9 and 60 degrees C at pH 10. The results of this study indicate that this novel C18 silica-based column represents a significant advancement in RPLC column technology with enhanced thermal and pH stability when compared to traditional bonded phase silica columns.