Development,validation, and application of an LC–MS/MS method for mitragynine and 7‐hydroxymitragynine analysis in hair

The entire scalp hair of a self‐declared Kratom consumer of 3 grams per day was acquired during an ethical committee approved study. As no values of the concentration in hair of the two Kratom alkaloids mitragynine or 7‐hydroxymitragynine were found in the literature, an already established method for the analysis of benzodiazepines/z‐substances was extended for the detection of mitragynine and 7‐hydroxymitragynine with LC–MS/MS, and successfully validated. The limits of detection and quantification for mitragynine were 2 pg/mg and 4 pg/mg, respectively. Those of 7‐hydroxymitragynine were 20 pg/mg and 30 pg/mg, respectively. The method was applied to the entire scalp hair, divided in 91 regions, of the study participant. A narrow mitragynine concentration distribution with values between 1054 pg/mg and 2244 ng/mg (mean 1517 ng/mg) and no clear scalp region associated distribution pattern was obtained. 7‐Hydroxymitragynine was not detected in any hair sample. After validation, the method was established as routine and subsequently 300 samples (mainly abstinence controls for drugs of abuse) were analyzed, allowing the investigation of the prevalence of Kratom consumption in our population. None of the analyzed routine hair samples were positive for mitragynine or 7‐hydroxymitragynine, providing no evidence that Kratom consumption is prevalent in the investigated population.     

Drug testing for mitragynine and kratom: Analytical challenges and medico-legal considerations

Mitragyna speciosa, known as kratom, is a tropical tree native to Southeast Asia that has long been used to increase energy and in traditional medicine. Kratom leaves contain several indole alkaloids including mitragynine, mitraciliatine, speciogynine, and speciociliatine, which have the same molecular formula and connectivity, but different spatial arrangements (i.e., diastereomers). A routine liquid-chromatographic–high-resolution mass-spectrometric (LC-HRMS) multi-analyte method for addictive and herbal drugs in urine did not separate mitragynine from speciogynine and speciociliatine. Separation and individual measurement of the four diastereomers was possible with an improved LC method. All diastereomers were detected in 29 patient urine samples who tested positive for mitragynine with the routine method, albeit at variable absolute amounts and relative proportions. The presence of all diastereomers rather than individual substances indicated that they originated from the intake of kratom (i.e., plant material). Speciociliatine dominated in most samples (66%), whereas mitragynine and mitraciliatine were the highest in 17% each. A kratom product (powdered plant material) marketed in Sweden contained all diastereomers with mitragynine showing the highest level. In Sweden, there are signs of an increasing use of kratom in society, based on the results from drug testing, the number of poisons center consultations on intoxications, and customs seizure statistics. Because there may be health risks associated with kratom use, including dependence, serious adverse reactions, and death, analytical methods should be able to identify and quantify all diastereomers. In Sweden, this is important from a legal perspective, as only mitragynine is classified, whereas the other three diastereomers, and kratom (plant material), are not.     

Ephedra alkaloid contents of Chinese herbal formulae sold in Taiwan

Consumption of Ephedra alkaloids is prohibited in‐competition by the World Anti‐Doping Agency (WADA). In Taiwan, colds are often treated with Chinese herbal formulae containing Herba Ephedrae. We screened products sold in Taiwan and preliminarily assessed their relationships with WADA threshold violations. Fifty‐six concentrated powder products, including 19 Chinese herbal formulae that contained Herba Ephedrae, were collected. The content of Ephedra alkaloids, namely ephedrine (E), methylephedrine (ME), norpseudoephedrine (NPE; cathine), pseudoephedrine (PE), and norephedrine (NE; phenylpropanolamine), was determined using a validated high‐performance liquid chromatography method. The results revealed that the phenotypic indicators of the collected products, E/PE and E/total ratios, were 1.52–4.70 and 0.49–0.72, respectively, indicating that the Herba Ephedrae species in these products was probably E. sinica or E. equisetina, but not E. intermedia. The contents of E, ME, NPE, PE, and NE and the total alkaloid contents in the daily doses of the products were 0.45–34.97, 0.05–4.87, 0.04–3.61, 0.15–12.09, and 0.01–2.00 mg and 0.68–53.64 mg, respectively. The alkaloid contents followed a relatively consistent order (E > PE > ME ≈ NPE > NE), even for products from different manufacturers. We calculated that single doses of 50.0% and 3.6% of the products would result in the WADA thresholds of E and NPE being exceeded, respectively. Our data provide critical information for athletes and medical personnel, who should be wary of using complex Chinese herbal formulae in addition to over‐the‐counter products.     

Development of an immunochromatographic strip incorporating anti‐mitragynine monoclonal antibody conjugated to colloidal gold for kratom alkaloids detection

A lateral flow‐based immunochromatographic strip was developed for the rapid detection of mitragynine (MG), a dominant alkaloid found in the leaves of kratom. Monoclonal antibody (mAb) against MG (anti‐MG mAb) was conjugated to colloidal gold and used as a recognition probe. MG‐ovalbumin conjugate (MG‐OVA) and goat anti‐mouse IgG were immobilized on the strip to produce a test zone and control zone, respectively. Based on the principle of a competitive assay, MG in a test sample competed with MG‐OVA resident in the test zone to bind with colloidal gold‐anti‐MG mAb, resulting in an inverse relation of color intensity at the test zone and MG amount. The limit of detection (LOD) of the immunochromatographic strip is determined at 1 mg/mL of MG by visual assessment and 0.60 mg/mL by Image J analysis. The developed immunochromatographic strip can determine MG in kratom cocktails and kratom leaf samples. It could serve as a rapid and simple diagnostic kit for the detection of MG in kratom samples.     

Optimization and validation of simultaneous analyses of ecgonine,cocaine, and seven metabolites in human urine by gas chromatography–mass spectrometry using a one‐step solid‐phase extraction

The presence of ecgonine in urine has been proposed as an appropriate marker of cocaine use. Only a few methods have been published for their determination along with cocaine and the rest of its metabolites. Due to their high polarity and consequent solubility in water, these have low recoveries, which is why it is necessary to increase the sensitivity, by the formation of hydrochloric salts or multiderivatization of the analytes or by performing two solid‐phase extractions (SPEs), considerably increasing the time and cost of the analysis. This work describes a fast and fully validated procedure for the simultaneous detection and quantification of ecgonine, ecgonine‐methyl‐ester, benzoylecgonine, nor‐benzoylecgonine, m‐hydroxybenzoylecgonine, cocaethylene, cocaine, norcocaine, and norcocaethylene in human urine (500 μL) using one SPE and simple derivatization. Separation and quantification were achieved by gas chromatography–electron ionization–mass spectrometry (GC–EI–MS) in selected‐ion monitoring mode. Quantification was performed by the addition of deuterated analogs as internal standards. Calibration curves were linear in the adopted ranges, with determination coefficients higher than 0.99. The lower limits of quantification ranged from 2.5 to 10 ng/mL. The intra‐ and inter‐day precision, calculated in terms of relative standard deviation, were 1.2%–14.9% and 1.8%–17.9%, respectively. The accuracy, in terms of relative error, was within a ± 16.4% interval. Extraction efficiency ranged from 84% to 103%. Compared with existing methods, the procedure described herein is fast, since only one SPE is required, and cost‐effective. In addition, this method provides a high recovery for ecgonine, resulting in a better alternative to the previously published methods.     

A multi‐analyte approach to help in assessing the severity of acute poisonings – Development and validation of a fast LC–MS/MS quantification approach for 45 drugs and their relevant metabolites with one‐point calibration

Diagnosis, monitoring of the efficiency of detoxification, and estimating the prognosis of acute poisonings are important tasks in emergency toxicology. Comprehensive screening and quantification of relevant substances by gas chromatography–mass spectrometry (GC–MS) or liquid chromatography–mass spectrometry (LC–MS) help in assessing the severity of most acute poisonings. Turnaround time for such analyses must be short enough to impact on clinical decisions. Therefore, a multi‐analyte LC–MS/MS approach with a 5‐minute gradient was developed and validated for 45 drugs and their active metabolites as a complement to an existing GC–MS approach using the same liquid–liquid extraction. The determination ranges were defined by quality control samples of low and high, representing concentrations from low therapeutic to highly toxic levels. To shorten the turnaround time, one‐point calibration was used. Validation showed low matrix effects and ionization effects of co‐eluting analytes thanks to APCI source as well as sufficient recoveries, precisions, and selectivities. For accuracy, 32 of the 45 compounds fulfilled the criteria for quantification in lower therapeutic and 41 in overdosed and toxic concentrations, considering limits of ±30% deviation. The reuse of the processed calibrator for a period of 30 days was possible for 32 compounds, showing sufficient stability at 8°C. In addition, analysis of authentic blood samples showed the applicability and yielded drug levels, which were comparable to those determined by fully validated therapeutic drug monitoring methods. In conclusion, the present approach in combination with the GC–MS approach should provide sufficient support for clinical assessment of the severity of poisonings with 68 compounds in an acceptable turnaround time.     

Detection of in utero cannabis exposure by umbilical cord analysis

According to the 2014 National Survey on Drug Use and Health, 5.3% of pregnant women smoked marijuana in the past month. This prevalence is expected to increase as a growing number of states and countries are now considering legalization. Although the umbilical cord is becoming a useful objective tool to detect in utero drug exposure, currently data about analytical methods and its utility to detect cannabis exposure are scarce. The objective of this work was to develop a method for the determination of Δ⁹‐tetrahydrocannabinol (THC), 11‐hydroxyTHC (THC‐OH), 11‐nor‐9‐carboxy‐THC (THCCOOH), 8‐β‐11‐dihydroxyTHC (THC‐diOH), THC and THCCOOH glucuronides, and cannabidiol (CBD) in the umbilical cord by liquid chromatography–tandem mass spectrometry (LC–MS/MS) with dual ionization source. Umbilical cord samples (0.5 g) were homogenized in methanol and extracted by solid‐phase extraction. Reversed‐phase chromatographic separation was performed in 14 minutes, and 2 transitions per analyte were monitored in multiple reaction monitoring mode. Method validation included linearity (1–10 to 20–200 ng/g), precision (4.1%–23.4%), accuracy (87.5%–111.4%), matrix effect (‐54.8% to ‐5.8%), extraction efficiency (25%–45.6%), limits of detection and quantification (1–10 ng/g), and endogenous (n = 5) or exogenous interferences (not detected). The method was applied to 13 authentic samples from cannabis‐exposed newborns, which meconium samples had tested positive for cannabis. Twelve cord specimens tested positive for THCCOOH‐glucuronide (1.6–19.1 ng/g). We developed and validated a specific and sensitive method for the simultaneous determination of THC, its metabolites, including THC and THCCOOH glucuronides, and CBD in umbilical cord samples by LC–MS/MS. The analysis of authentic samples showed the usefulness of umbilical cord to detect cannabis in utero exposure.     

Analytical quantification,intoxication case series,and pharmacological mechanism of action for N‐ethylnorpentylone (N‐ethylpentylone or ephylone)

Synthetic cathinones continue to proliferate in clandestine drug markets worldwide. N‐ethylnorpentylone (also known as N‐ethylpentylone or ephylone) is a popular emergent cathinone, yet little information is available about its toxicology and pharmacology. Here we characterize the analytical quantification, clinical presentation, and pharmacological mechanism of action for N‐ethylnorpentylone. Liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) was used to quantify N‐ethylnorpentylone in blood obtained from human cases. Clinical features exhibited by the intoxicated individuals are described. The activity of N‐ethylnorpentylone at plasma membrane transporters for dopamine (DAT), norepinephrine (NET) and 5‐HT (SERT) was assessed using in vitro assays measuring uptake inhibition and evoked release of [³H] neurotransmitters in rat brain synaptosomes. Our LC–MS/MS method assayed N‐ethylnorpentylone concentrations with limits of detection and quantification of 1 and 5 ng/mL, respectively. Quantitation was linear from 5 to 500 ng/mL, and the method displayed specificity and reproducibility. Circulating concentrations of N‐ethylnorpentylone ranged from 7 to 170 ng/mL in clinical cases, and the associated symptoms included palpitations, tachycardia, agitation, hallucinations, coma and death. N‐Ethylnorpentylone was a potent inhibitor at DAT (IC₅₀ = 37 nM), NET (IC₅₀ = 105 nM) and SERT (IC₅₀ = 383 nM) but displayed no transporter releasing activity. We present a validated method for quantifying N‐ethylnorpentylone in human case work. The drug is a psychomotor stimulant capable of inducing serious cardiovascular and neurological side‐effects which can be fatal. In vitro findings indicate that N‐ethylnorpentylone exerts its effects by potent blockade of DAT and NET, thereby elevating extracellular levels of dopamine and norepinephrine in the brain and periphery.     

PTCA (1H‐pyrrole‐2,3,5‐tricarboxylic acid) as a marker for oxidative hair treatment

Hair analysis for the assessment of alcohol or drug abstinence has become a routine procedure in forensic toxicology. Hair coloration leading to loss of incorporated xenobiotics and to false negative results has turned out to be a major problem. Currently only colored extracts provide hints of manipulations but not bleaching. A liquid chromatographic–tandem mass spectrometric (LC–MS/MS) method was developed and validated to determine 1H‐pyrrole‐2,3,5‐tricarboxylic acid (PTCA), a major oxidation product of melanin. PTCA was determined in natural hair samples (n = 21) after treatment with 3% hydrogen peroxide (H₂O₂) for 30 or 40 minutes with concentrations up to 12% for 40 minutes. In another series, 12 natural hair samples were submitted to different coloration procedures (henna, tinting, semi‐permanent and permanent dyeing, bleaching) and the changes in PTCA content were determined. A significant increase in the PTCA content was found for both incubation times and increasing H₂O₂ concentrations. Coloration with henna or tinting had no influence on PTCA levels detected, but a significant increase was observed after semi‐permanent and permanent dyeing and bleaching. As PTCA concentrations in natural hair were found to be in a range of <2.1–16.4 ng/mg (8.4 ± 3.8 ng/mg, mean ± SD, n = 33), a cut‐off of 20 ng/mg is recommended for the distinction between natural vs. excessively oxidized hair. In case of naturally low melanin content (light‐blond or white hair), no marked increase in PTCA may occur. The present study demonstrated that PTCA is formed during oxidative treatment of melanin in hair, which can be used to detect previous hair coloration including oxidation.     

Ultra‐fast LC–MS/MS in therapeutic drug monitoring: Quantification of clozapine and norclozapine in human plasma

A novel approach to high‐throughput, targeted liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis has been developed. A single chromatographic system can be used for the analysis of a range of 20 drugs and metabolites with a total analysis time of 36 s (one 96‐well plate of prepared samples per hour). To demonstrate the applicability of this approach to quantitative analysis, a method has been validated for the therapeutic drug monitoring of clozapine and norclozapine following automated extraction from human plasma. Chromatographic retention times were 11.4 and 12.4 s for norclozapine and clozapine, respectively (for both analytes the chromatographic peak width was less than 1 s). Comparison with a conventional LC–MS/MS method (5 min analysis time) showed excellent agreement. This new approach offers analysis times more akin to flow‐injection analysis, but is likely to be more widely applicable because of chromatographic resolution from residual matrix components and isobaric interferences.     

Detection of Δ9 THC in oral fluid following vaporized cannabis with varied cannabidiol (CBD) content: An evaluation of two point‐of‐collection testing devices

Point‐of‐collection testing (POCT) for Δ⁹‐tetrahydrocannabinol (THC) in oral fluid is increasingly used to detect driving under the influence of cannabis (DUIC). However, previous studies have questioned the reliability and accuracy of two commonly used POCT devices, the Securetec DrugWipe^® 5 s (DW5s) and Dräger DrugTest^® 5000 (DT5000). In the current placebo controlled, double‐blind, crossover study we used liquid chromatography‐tandem mass spectrometry (LC–MS/MS) to accurately quantify cannabinoid concentrations in the oral fluid of 14 participants at various timepoints (10, 60, 120, and 180 minutes) following vaporization of 125 mg of THC‐dominant (11% THC; <1% CBD), THC/CBD equivalent (11% THC; 11% CBD) and placebo (<1% THC; <1% CBD) cannabis. At each timepoint, oral fluid was also screened using the DW5s (10 ng/mL THC cut‐off) and DT5000 (10 ng/mL THC cut‐off). LC–MS/MS analysis showed peak oral fluid THC concentrations at the 10 minute timepoint with a rapid decline thereafter. This trajectory did not differ with THC dominant and THC/CBD equivalent cannabis. With a 10 ng/mL confirmatory cut‐off, 5% of DW5s test results were false positives and 16% false negatives. For the DT5000, 10% of test results were false positives and 9% false negatives. Neither the DW5s nor the DT5000 demonstrated the recommended >80% sensitivity, specificity and accuracy. Accuracy was lowest at 60 minutes, when THC concentrations were often close to the screening cut‐off (10 ng/mL). POCT devices can be useful tools in detecting recent cannabis use; however, limitations should be noted, and confirmatory LC–MS/MS quantification of results is strongly advisable.     

Rapid assessment of the illegal presence of 1,3‐dimethylamylamine (DMAA) in sports nutrition and dietary supplements using 1H NMR spectroscopy

1,3‐Dimethylamylamine (DMAA) is a stimulant that can be found in pre‐workout sports nutrition and dietary supplements. This practice is illegal because DMAA is not a safe food ingredient but rather an unapproved medicinal compound due to its pharmacological action. In order to determine the DMAA content in such products, a nuclear magnetic resonance (NMR) spectroscopic method was developed and validated (DMAA was quantified as DMAA‐HCl). For quantification, the collective integral from two of the methyl groups of the molecule in the range δ 0.92–0.84 ppm was used. The method was linear over the examined range of 1–21 g/kg (R² = 0.9937). The recoveries from spiked concentrations (0.1–6 g/kg) ranged between 85% and 105% (96% on average), with a relative standard deviation (RSD) of 1% for an authentic sample. The detection limit was 0.03 g/kg and the quantification limit was 0.08 g/kg (calculated for 75 mg sample weight). The actual DMAA‐HCl content in the sample was quantified using calibration curves (external standardization) or 3,5‐dinitrobenzoic acid as single‐point internal standard. The developed NMR methodology was applied for the analysis of 16 products, from which 9 samples were found positive (the DMAA‐HCl concentration varied between 3.1 g/kg and 415 g/kg). The method can be recommended for routine use in food testing, customs or doping control laboratories. Copyright © 2014 John Wiley & Sons, Ltd.     

Kratom policy: The challenge of balancing therapeutic potential with public safety

Kratom (Mitragyna speciosa) is a tree-like plant indigenous to Southeast Asia. Its leaves, and the teas brewed from them have long been used by people in that region to stave off fatigue and to manage pain and opioid withdrawal. Evidence suggests kratom is being increasingly used by people in the United States and Europe for the self-management of opioid withdrawal and treatment of pain. Recent studies have confirmed that kratom and its chemical constituents have potentially useful pharmacological actions. However, there have also been increasing numbers of reports of adverse effects resulting from use of kratom products. In August 2016, the US Drug Enforcement Administration announced plans to classify kratom and its mitragynine constituents as Schedule I Controlled Substances, a move that triggered a massive response from pro-kratom advocates. The debate regarding the risks, and benefits and safety of kratom continues to intensify. Kratom proponents tout kratom as a safer and less addictive alternative to opioids for the management of pain and opioid addiction. The anti-kratom faction argues that kratom, itself, is a dangerous and addictive drug that ought to be banned. Given the widespread use of kratom and the extensive media attention it is receiving, it is important for physicians, scientists and policy makers to be knowledgeable about the subject. The purpose of this commentary is to update readers about recent developments and controversies in this rapidly evolving area. All of the authors are engaged in various aspects of kratom research and it is our intention to provide a fair and balanced overview that can form the basis for informed decisions on kratom policy. Our conclusions from these analyses are: (a) User reports and results of preclinical studies in animals strongly suggest that kratom and its main constituent alkaloid, mitragynine may have useful activity in alleviating pain and managing symptoms of opioid withdrawal, even though well-controlled clinical trials have yet to be done. (b) Even though kratom lacks many of the toxicities of classic opioids, there are legitimate concerns about the safety and lack of quality control of purported “kratom” products that are being sold in the US. (c) The issues regarding the safety and efficacy of kratom and its mitragynine constituent can only be resolved by additional research. Classification of the Mitragyna alkaloids as Schedule I controlled substances would substantially impede this important research on kratom.     

Alkaloid profiling of herbal drugs using high resolution mass spectrometry

Herbal infusions are consumed worldwide thanks to their “natural” beneficial effects, also due to the presence of alkaloids, although these compounds can have poisonous effects. A method combining online solid‐phase purification with high resolution mass spectrometry was used to define the alkaloid profiles of 117 herbs and 7 commercial blends. Forty‐one alkaloids were quantified in reference to analytical standards, while the presence of a further 116 was confirmed based on accurate mass, retention time, and fragmentation profile. The targeted study showed that 52% of herbs and 42% of commercial blends contained at least one alkaloid. Pyrrolizidines were the most commonly present (26% of samples), with concentrations generally ranging from the quantification limit to roughly 100 μg kg^?1. Moreover, a homemade infusion was studied, finding on average 45% and 6% lower extraction for pyrrolizidine and steroidal alkaloids, respectively. Nevertheless, the migration of pyrrolizidines was confirmed. The study confirmed the frequent presence, natural or accidental, of alkaloids in commercial infusion herbs, highlighting the urgent need for routine and accurate controls.     

High‐throughput doping control analysis of 28 amphetamine‐type stimulants in equine plasma using hydrophilic interaction liquid chromatography–tandem mass spectrometry

A hydrophilic interaction liquid chromatography–tandem mass spectrometry method (HILIC–MS/MS) was developed for the simultaneous determination of 28 amphetamine‐type stimulants (ATSs) in equine plasma for doping control analysis. In this method, stimulants were recovered from equine plasma by liquid–liquid extraction (LLE) at pH 9.5 using methyl tert‐butyl ether and detected on a Thermo Finnigan triple quadrupole mass spectrometer operating in positive‐ion mode electrospray ionization. All stimulants were eluted within 7 minutes and baseline separation was achieved for isomeric and isobaric compounds using HILIC chromatography. Extraction efficiency was greater than 80% and matrix effect was acceptable for most stimulants. The limit of detection (LOD) was in the range of 10–50 pg/mL and the lower limit of quantification (LLOQ) was in the range of 50–100 pg/mL. Quadratic regression was employed for quantification and the dynamic range of quantification was 50–10000 pg/mL. Confirmatory analysis criteria were established using product ion ratios and retention time. The limit of confirmation (LOC) was in the range of 20–100 pg/mL. Stability study results indicated that some stimulants were unstable in equine plasma at room temperature and 4°C. However, all the stimulants studied were stable at ?20°C and ? 80°C for the 6 month study period.     

Effects of Uncaria tomentosa total alkaloid and its components on experimental amnesia in mice: elucidation using the passive avoidance test

The effects of Uncaria tomentosa total alkaloid and its oxindole alkaloid components, uncarine E, uncarine C, mitraphylline, rhynchophylline and isorhynchophylline, on the impairment of retention performance caused by amnesic drugs were investigated using a step-down-type passive avoidance test in mice. In this test, the retention performance of animals treated with the amnesic and test drugs before training was assessed 24 h after training. Uncaria tomentosa total alkaloid (10-20 mg kg(-1), i.p.) and the alkaloid components (10-40 mg kg(-1), i.p.), as well as the muscarinic receptor agonist oxotremorine (0.01 mg kg(-1), i.p.), significantly attenuated the deficit in retention performance induced by the muscarinic receptor antagonist scopolamine (3 mg kg(-1), i.p.). The effective doses of uncarine C and mitraphylline were larger than those of other alkaloid components. Uncarine E (20 mg kg(-1), i.p.) also blocked the impairment of passive avoidance performance caused by the nicotinic receptor antagonist mecamylamine (15 mg kg(-1), i.p.) and the N-methyl-D-aspartate (NMDA) receptor antagonist (+/-)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP; 7.5 mg kg(-1), i.p.), but it failed to affect the deficit caused by the benzodiazepine receptor agonist diazepam (2 mg kg(-1), i.p.). Rhynchophylline significantly reduced the mecamylamine-induced deficit in passive avoidance behaviour, but it failed to attenuate the effects of CPP and diazepam. These results suggest that Uncaria tomentosa total alkaloids exert a beneficial effect on memory impairment induced by the dysfunction of cholinergic systems in the brain and that the effect of the total alkaloids is partly attributed to the oxindole alkaloids tested. Moreover, these findings raised the possibility that the glutamatergic systems are implicated in the anti-amnesic effect of uncarine E.     

UHPLC–MS/MS quantitation of porphyroxine in opium and application of porphyroxine‐acetylated products as signature markers for heroin

Porphyroxine, a trace alkaloid in opium, was identified in the early 1800s and isolated/characterized in the 1960s. Recently, two significant porphyroxine‐related byproducts found in the acidic and neutral extracts of illicit heroin were characterized by this laboratory as the N‐acetyl‐O¹⁴‐desmethyl‐epi‐porphyroxine ( B ) and N,O⁸‐diacetyl‐O¹⁴‐desmethyl‐epi‐porphyroxine ( C ). The prevalence of the B and C compounds has been consistent in the following order of abundance for the thousands of authentic heroin samples analyzed: Southwest Asia (SWA) > South America (SA) > Southeast Asia (SEA) > Mexico (MEX). In this research, a rapid and efficient ultra‐high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method was developed to determine the content of porphyroxine and five primary alkaloids (morphine, codeine, thebaine, noscapine, and papaverine) in opium after extraction with methanol/water (50/50). The method was validated in terms of linearity, accuracy, recovery, and precision for porphyroxine. The limit of quantitation (LOQ) for porphyroxine was 2.5 ng/mL. The developed method was successfully applied to a total of 114 authentic opium samples from the major poppy‐growing regions. The amount of porphyroxine was determined at the level of part per thousand (‰) and the relative concentrations to morphine were in the range of 1x10^?4 and 1x10^?2 with an order of SWA > SEA, SA > MEX for its average abundance, which is consistent with the order of the average abundance of its acetylated products ( B , C ) in illicit heroin. This study reveals the significance of porphyroxine and its acylated compounds in classifying heroin and opium samples to major geographical regions of production.     

Development and validation of a fast and simple multi‐analyte procedure for quantification of 40 drugs relevant to emergency toxicology using GC‐MS and one‐point calibration

Diagnosis and prognosis of poisonings should be confirmed by comprehensive screening and reliable quantification of xenobiotics, for example by gas chromatography–mass spectrometry (GC‐MS) or liquid chromatography‐mass spectrometry (LC‐MS). The turnaround time should be short enough to have an impact on clinical decisions. In emergency toxicology, quantification using full‐scan acquisition is preferable because this allows screening and quantification of expected and unexpected drugs in one run. Therefore, a multi‐analyte full‐scan GC‐MS approach was developed and validated with liquid‐liquid extraction and one‐point calibration for quantification of 40 drugs relevant to emergency toxicology. Validation showed that 36 drugs could be determined quickly, accurately, and reliably in the range of upper therapeutic to toxic concentrations. Daily one‐point calibration with calibrators stored for up to four weeks reduced workload and turn‐around time to less than 1 h. In summary, the multi‐analyte approach with simple liquid‐liquid extraction, GC‐MS identification, and quantification over fast one‐point calibration could successfully be applied to proficiency tests and real case samples. Copyright © 2013 John Wiley & Sons, Ltd.     

草乌花及其煎煮液中二萜生物碱的电喷雾串联质谱研究草乌花及其煎煮液中二萜生物碱的电喷雾串联质谱研究

目的 对草乌花及其煎煮液中的二萜生物碱进行定性分析,说明煎煮前后化学成分发生的变化。 方法用注射泵自动进样,电喷雾离子阱串联质谱直接分析草乌花及其煎煮液中生物碱混合物。结果 在生草乌花中发现3个新生物碱,草乌花煎煮后其中的双酯型生物碱和三酯型生物碱都发生水解,前者水解为苯甲酰乌头原碱和乌头原碱类生物碱,后者水解为3-乙酰-乌头原碱类生物碱。结论 该法简便、快速、灵敏、特异性强,为乌头属植物煎煮液中的生物碱分析提供了新途径。     

Creatinine in urine – a method comparison

Drug screening in urine is widely applied in forensic toxicology. Contrary to blood analysis, excessive or reduced fluid intake can substantially alter the concentration of substances in urine. As a standard to detect urinary dilution, creatinine concentrations are analyzed. A sample with a concentration below 20 mg/dL is generally defined as too diluted to provide a valid result in abstinence control samples. This work investigates the potential of three different methods for the determination of creatinine concentrations in urine samples: A ZIC®‐HILIC‐based liquid chromatography–tandem mass spectrometry (LC–MS/MS) method, a spectrophotometric method on an AU 480 chemistry system, and a portable, chemical‐reaction‐based, point‐of‐care testing device were compared by measuring 200 urine samples. When comparing the two laboratory methods, LC–MS/MS and spectrophotometry, a mean difference of 3.7 ± 14 mg/dL was found, indicating that the spectrophotometric method slightly overestimates the creatinine concentration. When comparing the LC–MS/MS method with the point‐of‐care testing device, a mean concentration difference within the calibration range for POCT (>20 mg/dL (excluding 16 samples) and <500 mg/dL (excluding 4 samples)) of 21 ± 37 mg/dL was found, indicating that the point‐of‐care testing device overestimates the measured creatinine concentration. A point‐of‐care testing device as used during this study can provide valuable information for on‐site analysis. However, reported concentrations above 500 mg/dL should be further evaluated, for example by dilution of the sample. Copyright © 2017 John Wiley & Sons, Ltd.