The reliable identification of amphetamine in degraded biological samples is often complicated by the presence of putrefactive amines, such as phenethylamine, which exhibit similar physicochemical properties and can interfere with chromatographic separation. This study aimed to systematically evaluate the conditions of gas chromatography–mass spectrometry (GC–MS), including derivatization methods and analytical column selection, to achieve effective separation and identification of amphetamine in the presence of phenethylamine. Various derivatization approaches, including acylation, alkoxycarbonylation, silylation, and pentafluorobenzylation, were examined in combination with non-polar, slightly polar, and semi-polar GC columns. The chromatographic behavior of amphetamine and phenethylamine, including the retention time, peak shape, and formation of mono- or di-substituted derivatives, was systematically assessed to optimize separation and mass spectral identification. Alkoxycarbonylation, silylation, and alkylation produced overlapping peaks, mixed derivatives, or unreacted compounds, thereby limiting their reliable identification. Acyl derivatization allowed for effective separation using non-polar and semi-polar columns, with the non-polar column minimizing peak tailing that is commonly caused by high phenethylamine concentrations. Differences in the elution order were observed between column types, and semi-polar columns provided satisfactory chromatographic resolution and high-quality mass spectra, consistent with spectral databases. Successful GC–MS identification of amphetamine in putrefied samples requires careful selection of the derivatization chemistry and GC column polarity. Acyl derivatization combined with an appropriate column choice enables reliable separation from phenethylamine, improves spectral quality, and enhances the evidential value of forensic analysis.

Supported liquid extraction (SLE) is commonly used to avoid emulsion formation; however, it takes a long time to concentrate the eluate and it is difficult to use in multiple samples. To solve these problems, automation of SLE has been previously reported, but no paper has reported the automation of the entire pretreatment operation in one apparatus. Therefore, we applied ATLAS-LEXT, an automated pretreatment apparatus developed to fully automate liquid-liquid extraction (LLE), to SLE and investigated the full automation of the pretreatment operation in a blood drug analysis. Blood samples containing 18 benzodiazepines and 16 other psychotropic drugs were pretreated with ATLAS-LEXT and analyzed using liquid chromatography/mass spectrometry (LC/MS). For all benzodiazepines tested, the calibration curves showed good linearity within the range of 10-500 ng/mL, and the recovery rate was ≥79%. For the other psychotropic drugs, the calibration curve showed good linearity within the range of 10-500 ng/mL, although the recovery rate was low for some analytes. In conclusion, the present study suggests that ATLAS-LEXT is useful for the pretreatment of blood samples for drug analysis using SLE, which has been impossible to fully be automated until now.

Purpose Cannabis is the most abused drug in the world and molecular biology techniques for the analysis of cannabis have attracted attention in recent years. We report a case of three persons conspiring to possess cannabis-like leaf pieces for profit. We present the results of DNA testing by a simple kit on suspected cannabis samples with exceptionally shaped leaves. Methods DNA was obtained from the samples of cannabis leaves by a simple and rapid procedure using a cannabis DNA detection kit or commercially available purified plant DNA extraction kit. Undiluted or diluted DNA solution was used as templates for amplification of cannabis-specific sequences by triplex PCR. The presence or absence of PCR amplicon was visually observed by a DNA chromatography detection system to determine whether the sample was cannabis or not. Results When the DNA was extracted by the simple procedure and used without dilution, PCR failed to identify cannabis samples. However, when purified DNA was used, PCR amplification was successful in identifying all cannabis samples. Notably, in case of DNA extracted by the simple procedure, 10 to 50-fold dilution of the solution reduced PCR inhibition and PCR amplification was successful. Conclusions Although the quality of the DNA used as PCR template needs to be considered, simple DNA testing with a kit is very effective for identifying suspected cannabis samples with exceptional morphology.

Recently, various synthetic cannabinoid (SC) compounds that have been slightly modified at the functional groups have been identified in Japan. However, the structural elucidation of these new compounds using conventional approaches such as gas chromatography–electron impact–mass spectrometry (GC-EI-MS) is difficult. As such, indole and indazole SCs were scanned using GC-MS-EI, positive GC–chemical ionization (CI)–MS, and negative GC–chemical ionization–MS, allowing for efficient structural elucidation of unknown SC compounds. Pure substances have been employed for the study.

A number of N-alkyl indole or indazole-3-carbonyl analogs, with modified chemical structures, are distributed throughout the world as synthetic cannabinoids. Like synthetic cannabinoids, cathinone analogs are also abused and cause serious problems worldwide. Acute deaths caused by overdoses of these drugs have been reported. Various analytical methods that can cope with the rapid changes in chemical structures are required for routine analysis and screening of these drugs in seized and biological materials for forensic and clinical purposes. Although many chromatographic methods to analyze each drug have been published, there are only a few articles summarizing these analytical methods. This review presents the various colorimetric detections, immunochemical assays, gas chromatographic-mass spectrometric methods, and liquid chromatographic-mass spectrometric methods proposed for the analysis of synthetic cannabinoids and cathinones.

We report the time-course profile of urinary excretion of α-pyrrolidinovalerophenone (α-PVP) and α-pyrrolidinobutiophenone (α-PBP) after intravenous injection in a human. The subject was a man aged in his forties, who intravenously injected unknown amounts of so-called unregulated drugs with the intention to commit suicide. The urinary concentrations of the drugs were analyzed by MonoSpin® extraction and gas chromatography–mass spectrometry. α-PVP, α-PBP, and their 2′-oxo metabolites of these compounds were identified in the urine. The highest concentration of α-PVP and α-PBP in urine was 1.2 and 1.6 μg/ml, respectively. The excretion half-lives of α-PVP and α-PBP were calculated to be 22 and 11 h, respectively. To our knowledge, this is the first report on the urinary excretion profiles of α-PVP and α-PBP in a human.

A monolithic spin column was developed for the extraction of analytes from biological materials. This column was constructed by packing a monolithic silica disk into a spin column. Sample loading, washing, and elution of the target drugs were accomplished simply by centrifugation of the column. Opiates and benzodiazepines are abused throughout the world. Identification and quantification of these drugs is very important to solve crimes or the cause of death. Three opiates (morphine, codeine, and dihydrocodeine) were extracted from urine and serum by using the column. After conversion to trimethylsilyl derivatives of the opiates by vigorous mixing with the derivatizing reagent, the solution was subjected to GC/MS. A linear curve was observed for opiates from 10 to 2500 ng/mL in urine and 5 to 1200 ng/mL in serum, respectively (correlation coefficient >0.996). For benzodiazepines, the hydroxyl metabolites of triazolam and etizolam were extracted from urine using the column, and the eluate was directly analyzed by HPLC/MS without evaporation. The LOD values were at the ppb level, with RSD values lower than 15%. The proposed methods were successfully applied to clinical and forensic cases, and good agreement of results was obtained compared to conventional methods.

A method coupling spin column extraction with gas chromatography-mass spectrometry was developed for the simultaneous extraction of acidic and basic drugs from urine. Benzodiazepines, local anaesthetics, antidepressants, and barbiturates were used as model drugs. Sample loading, washing, and elution of the target drugs were accomplished by centrifugation of the column. In this study, mixed-mode monolithic silica bonded with a C18 reversed-phase and a strong cation exchange phase was packed in a spin column. The pH of a urine sample (0.2 mL) was adjusted to 3 and the analytes adsorbed onto the column were eluted with 0.1 mL of MeOH containing 2% NH(3); all the tested drugs were simultaneously extracted from urine. The recovery of the tested drugs was 65-123%. Up to a concentration of 2500 ng/mL of the target drugs in urine, a linear curve was observed (r(2)>0.996). The intra- and interday RSDs at three different concentrations in urine were 2.1-14.7%. For RSDs lower than 15%, the limits of detection were 1-25 ng/mL. The proposed method was successfully applied for clinical and forensic cases and the results thus obtained were in good agreement with those obtained by conventional methods.

We present a method based on monolitic spin column extraction and gas chromatography–mass spectrometry as an analytical method for screening diquat (DQ), paraquat (PQ), and fenitrothion in serum and urine. This method is useful for clinical and forensic toxicological analyses. Recovery of DQ, PQ, and fenitrothion from serum and urine, spiked at concentrations between 0.1, 2.5, 20, and 45 μg/ml, ranged from 51.3% to 106.1%. Relative standard deviation percentages were between 3.3% and 14.8%. Detection and quantitation limits for serum and urine were 0.025 and 0.05 μg/ml, respectively, for DQ, 0.1 and 0.1 μg/ml, respectively, for PQ, and 0.025 and 0.05 μg/ml, respectively, for fenitrothion. Therefore, these compounds can be detected and quantified in the case of acute poisoning.

A number of analogues of phenethylamine and tryptamine, which are prepared by modification of the chemical structures, are being developed for circulation on the black market. Often called “designer drugs,” they are abused in many countries, and cause serious social problems in many parts of the world. Acute deaths have been reported after overdoses of designer drugs. Various methods are required for screening and routine analysis of designer drugs in biological materials for forensic and clinical purposes. Many sample preparation and chromatographic methods for analysis of these drugs in biological materials and seized items have been published. This review presents various colorimetric detections, gas chromatographic (GC)–mass spectrometric, and liquid chromatographic (LC)–mass spectrometric methods proposed for designer drug analyses. Basic information on extractions, derivatizations, GC columns, LC columns, detection limits, and linear ranges is also summarized.

A rapid, specific, and sensitive method for the simultaneous quantitation of organophosphates (fenitrothion (MEP), malathion, and phenthoate (PAP)), glufosinate (GLUF), and glyphosate (GLYP) in human serum and urine by gas chromatography-mass spectrometry (GC-MS) has been validated. All of the targeted compounds together with the internal standard were extracted from the serum and urine using a mix-mode TiO-C^1_8 monolithic spin column. The recovery of organophosphates from serum and urine ranged from 12.7 to 49.5%. The recovery of GLUF and GLYP from serum and urine ranged from 1.9 to 7.9%. The intra- and inter-accuracy and precision (expressed as relative standard deviation, %RSD) were within 96.7 - 107.7% and 4.0 - 13.8%, respectively. The detection and quantitation limits for serum and urine were 0.1 and 0.1 µg/ml, respectively, for organophosphates, 0.1 and 0.5 µg/ml, respectively for GLUF and GLYP. The method had linear calibration curves ranging from 0.1 to 25.0 µg/ml for organophosphates and 0.5 - 100.0 µg/ml for GLUF, and GLYP. The validated method was successfully applied to a clinical GLYP poisoning case.

Monolith was first used as a material for chromatographic separation two decades ago and solid-phase extraction over 10 years, and since then, separation science has undergone a dramatic change owing to advancements in analytical technology. Recently, monolith has been modified to suit various devices for the extraction and enrichment of analytes in any matrices of environmental, food, and biological analyses. This approach has contributed to miniaturization and automation for sample preparation, and it can reduce the time and cost requirements of sample preparation. Recently, numerous applications have been demonstrated for online and inline preconcentration coupled with monolith, and many kinds of devices have been designed and developed for offline devices. In this review, these applications and devices are listed and discussed in reference to other fields.

During the last one or two decades, monolithic polymers and silica have been developed for use as a new separation material or as a solid phase extraction sorbent and the applications with monoliths have been dramatically increased to separate the analytes by HPLC and to extract the analytes by solid phase extraction. The structure of monoliths differs from those of conventional particle materials. Specifically, the porosity of monoliths is over 80% and is larger than that of silica particle materials; therefore, columns packed with monoliths provide fast and high throughput analysis. Furthermore, high flow analysis is possible using monoliths because they produce very low back pressure. The applications of HPLC using monoliths for separation of bio-active compounds have gradually increased. In this review, these applications are summarized so that researchers can be introduced to the advantages of using monolithic silica. In addition, monolithic materials have the potential to be used as conventional solid phase extraction sorbents for the extraction of analytes in a sample matrix. The analytes in the sorbent can be eluted with a small volume because the monolith has wider surface area for each unit volume than those in other silicas or polymers and the required volume of the sorbents to extract the analytes is smaller in comparison with the conventional methods. To utilize these advantages of monoliths, new devices have been developed. In this review, the applications of monolithic silica for the extraction of drugs and medicines in biological materials are summarized.

Normal phase liquid chromatography (NPLC) has been widely used for the separation of polar compounds. In NPLC, a non-aqueous organic solution is used as the mobile phase. It is difficult to separate polar compounds in the mobile phase. Therefore, NPLC finds limited application in the separation of polar compounds from biological materials. Polar compounds are poorly retained in a reversed phase (RP) column, even if a highly aqueous mobile phase is used. For RPLC-mass spectrometry with electrospray ionization, poor analyte on-column retention may result in detrimental matrix effects and high water content. The mobile phase is also not conducive to achieving good spray conditions.Hydrophilic interaction LC (HILIC) is a relatively new separation technique used for polar and water-soluble compounds. HILIC with bare silica as the stationary phase and a low-aqueous/high-organic solution as the mobile phase has been used for the determination of polar compounds in biological materials. Neutral and hydrophobic endogenous interferences present in biological materials will not be retained on the column.Marine toxins and pharmaceuticals such as tetrodotoxin, saxitoxin, and antibiotics give rise to many polar compounds that act as contaminants. So far, RPLC and/or ion-pairing chromatography have been examined in order to separate and identify polar compounds in biological materials; however, some problems remain unsolved. The high-aqueous mobile phase or ion-paring reagents were ionized in the mass spectrometer. To overcome this problem, we have investigated the use of HILIC for separating polar compounds by using a low-aqueous/high-organic mobile phase. This article summarizes applications to separate marine toxins and pharmaceuticals that cause poisoning. We also describe HILIC's potential to determine toxins and pharmaceuticals in biological materials.

A rapid gas chromatography-mass spectrometry (GC-MS) method was developed and validated for the analysis of eperisone in serum using monolithic spin-column extraction. The linear concentration range for eperisone was 2-2500 ng/ml. The limit of detection was found to be 0.5 ng/ml. The average extraction recovery range was 92.8-96.0%. The intra- and interday relative standard deviations (RSDs) of the concentrations were less than 12.6% and 12.5%, respectively. The accuracy of this method ranged from 95.0% to 98.3%. We successfully used this assay to analyze serum samples from an eperisone-overdose patient. Our method has some analytical advantages compared with previously reported gas chromatography (GC) and GC-MS methods, such as higher selectivity and sensitivity than GC with nitrogen-phosphorus detection and the avoidance of nonspecificity, the ability to use a smaller sample volume than that required for the GC-MS method, and a shorter sample preparation time than the previous solid-phase extraction (SPE) method.

A simple, sensitive, and specific method with gas chromatography-mass spectrometry was developed for simultaneous extraction and derivatization of amphetamines (APs) and 3,4-methylenedioxyamphetamines (MDAs) in human urine by using a monolithic silica spin column. All the procedures, such as sample loading, washing, and elution were performed by centrifugation. APs and MDAs in urine were adsorbed on the monolithic silica and derivatized with propyl chloroformate in the column. Methamphetamine-d(5) was used as an internal standard. The linear ranges were 0.01-5.0 microg mL(-1) for methamphetamine (MA) and 3,4-methylenedioxymethamphetamine (MDMA) and 0.02-5.0 microg mL(-1) for amphetamine (AP) and 3,4-methylenedioxyamphetamine (MDA) (coefficient of correlation > or = 0.995). The recovery of APs and MDAs in urine was 84-94%, and the relative standard deviation of the intra- and interday reproducibility for urine samples containing 0.1, 1.0, and 4.0 microg mL(-1) of APs and MDAs ranged from 1.4% to 13.6%. The lowest detection limit (signal-to-noise ratio > or = 3) in urine was 5 ng mL(-1) for MA and MDMA and 10 ng mL(-1) for AP and MDA. The proposed method can be used to perform simultaneous extraction and derivatization on spin columns that have been loaded with a small quantity of solvent by using centrifugation.

We describe and validate a gas chromatography-mass spectrometry (GC-MS) method for the simultaneous quantitative detection of nine cold medication compounds and bromoisovaleryl urea an over-the-counter cold medication, in human serum; the nine compounds are acetaminophen (APAP), codeine, dihydrocodeine, three ephedrines, ethenzamide, ibuprofen, and salicylic acid. After adding the internal standard, acetaminophen-d 4 (APAP-d 4), compounds were extracted from the serum samples in the monolithic spin column. The extracted samples were evaporated to dryness. The residues were derivatized with acetonitrile and N-methyl-N-(tert-butyldimethylsilyl) trifluoroacetamide + 1% tert-butyldimethylchlorosilane, which were subjected to GC-MS analysis. The limit of quantification (LOQ) was 0.005–0.1 μg mL−1. The calibration curves were linear (r 2 > 0.995) in the concentration range from the LOQ to 10 μg mL−1. The intra- and inter-day variations, determined by the measurement of quality control samples at three tested concentrations, showed acceptable values. The lower limit of detection was between 0.005 and 0.05 μg mL−1. Mean recoveries from the serum samples were between 2.5 and 73.8%. This procedure was applied in the toxicological analysis of an intoxicated patient who was responsible for a traffic accident.

To overcome the limitations of solid-phase extraction, we developed a device comprising a spin column packed with octadecyl silane-bonded monolithic silica for extracting amphetamines and methylenedioxyamphetamines from urine. Urine (0.5mL), buffer (0.4mL), and methoxyphenamine (internal standard) were directly put into the preactivated column. The column was centrifuged (3000rpm, 5min) for sample loading and washed. The adsorbed analytes were eluted and analyzed by high-performance liquid chromatography, without evaporation. The results were as follows: linear curves (drug concentrations of 0.2–20μg/mL); correlation coefficients >0.99; detection limit, 0.1μg/mL. The proposed method is not only useful for drugs from biological materials but also highly reproducible for the analysis of these drugs in urine.