Plasma and urine profiles of Δ9-tetrahydrocannabinol and its metabolites 11-hydroxy-Δ9-tetrahydrocannabinol and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol after cannabis smoking by male volunteers to estimate recent consumption by athletes

Since 2004, cannabis has been prohibited by the World Anti-Doping Agency for all sports competitions. In the years since then, about half of all positive doping cases in Switzerland have been related to cannabis consumption. In doping urine analysis, the target analyte is 11-nor-9-carboxy-Δ⁹-tetrahydrocannabinol (THC-COOH), the cutoff being 15 ng/mL. However, the wide urinary detection window of the long-term metabolite of Δ⁹-tetrahydrocannabinol (THC) does not allow a conclusion to be drawn regarding the time of consumption or the impact on the physical performance. The purpose of the present study on light cannabis smokers was to evaluate target analytes with shorter urinary excretion times. Twelve male volunteers smoked a cannabis cigarette standardized to 70 mg THC per cigarette. Plasma and urine were collected up to 8 h and 11 days, respectively. Total THC, 11-hydroxy-Δ⁹-tetrahydrocannabinol (THC-OH), and THC-COOH were determined after hydrolysis followed by solid-phase extraction and gas chromatography/mass spectrometry. The limits of quantitation were 0.1–1.0 ng/mL. Eight puffs delivered a mean THC dose of 45 mg. Plasma levels of total THC, THC-OH, and THC-COOH were measured in the ranges 0.2–59.1, 0.1–3.9, and 0.4–16.4 ng/mL, respectively. Peak concentrations were observed at 5, 5–20, and 20–180 min. Urine levels were measured in the ranges 0.1–1.3, 0.1–14.4, and 0.5–38.2 ng/mL, peaking at 2, 2, and 6–24 h, respectively. The times of the last detectable levels were 2–8, 6–96, and 48–120 h. Besides high to very high THC-COOH levels (245 ± 1,111 ng/mL), THC (3 ± 8 ng/mL) and THC-OH (51 ± 246 ng/mL) were found in 65 and 98% of cannabis-positive athletes’ urine samples, respectively. In conclusion, in addition to THC-COOH, the pharmacologically active THC and THC-OH should be used as target analytes for doping urine analysis. In the case of light cannabis use, this may allow the estimation of more recent consumption, probably influencing performance during competitions. However, it is not possible to discriminate the intention of cannabis use, i.e., for recreational or doping purposes. Additionally, pharmacokinetic data of female volunteers are needed to interpret cannabis-positive doping cases of female athletes.     

Cannabinoids and metabolites in expectorated oral fluid after 8 days of controlled around-the-clock oral THC administration

Oral fluid (OF) is an increasingly accepted matrix for drug testing programs, but questions remain about its usefulness for monitoring cannabinoids. Expectorated OF specimens (n = 360) were obtained from 10 adult daily cannabis smokers before, during, and after 37 20-mg oral Δ⁹-tetrahydrocannabinol (THC) doses over 9 days to characterize cannabinoid disposition in this matrix. Specimens were extracted and analyzed by gas chromatography–mass spectrometry with electron-impact ionization for THC, 11-hydroxy-THC, cannabidiol, and cannabinol, and negative chemical ionization for 11-nor-9-carboxy-THC (THCCOOH). Linear ranges for THC, 11-hydroxy-THC, and cannabidiol were 0.25–50 ng/mL; cannabinol 1–50 ng/mL; and THCCOOH 5–500 pg/mL. THCCOOH was the most prevalent analyte in 344 specimens (96.9%), with concentrations up to 1,390.3 pg/mL. 11-hydroxy-THC, cannabidiol, and cannabinol were detected in 1, 1, and 3 specimens, respectively. THC was detected in only 13.8% of specimens. The highest THC concentrations were obtained at admission (median 1.4 ng/mL, range 0.3–113.6) from previously self-administered smoked cannabis. A total of 2.5 and 3.7% of specimens were THC-positive at the recommended Substance Abuse and Mental Health Services Administration (2 ng/mL) and Driving Under the Influence of Drugs, Alcohol and Medicines (DRUID) (1 ng/mL) confirmation cutoffs, respectively. THC is currently the only analyte for monitoring cannabis exposure in OF; however, these data indicate chronic therapeutic oral THC administration and illicit oral THC use are unlikely to be identified with current guidelines. Measurement of THCCOOH may improve the detection and interpretation of OF cannabinoid tests and minimize the possibility of OF contamination from passive inhalation of cannabis smoke.     

Oral fluid/plasma cannabinoid ratios following controlled oral THC and smoked cannabis administration

Oral fluid (OF) is a valuable biological alternative for clinical and forensic drug testing. Evaluating OF to plasma (OF/P) cannabinoid ratios provides important pharmacokinetic data on the disposition of drug and factors influencing partition between matrices. Eleven chronic cannabis smokers resided on a closed research unit for 51 days. There were four 5-day sessions of 0, 30, 60, and 120 mg oral ?⁹-tetrahydrocannabinol (THC)/day followed by a five-puff smoked cannabis challenge on Day 5. Each session was separated by 9 days ad libitum cannabis smoking. OF and plasma specimens were analyzed for THC and metabolites. During ad libitum smoking, OF/P THC ratios were high (median, 6.1; range, 0.2–348.5) within 1 h after last smoking, decreasing to 0.1–20.7 (median, 2.1) by 13.0–17.1 h. OF/P THC ratios also decreased during 5-days oral THC dosing, and after the smoked cannabis challenge, median OF/P THC ratios decreased from 1.4 to 5.5 (0.04–245.6) at 0.25 h to 0.12 to 0.17 (0.04–5.1) at 10.5 h post-smoking. In other studies, longer exposure to more potent cannabis smoke and oromucosal cannabis spray was associated with increased OF/P THC peak ratios. Median OF/P 11-nor-9-carboxy-THC (THCCOOH) ratios were 0.3–2.5 (range, 0.1–14.7) ng/μg, much more consistent in various dosing conditions over time. OF/P THC, but not THCCOOH, ratios were significantly influenced by oral cavity contamination after smoking or oromucosal spray of cannabinoid products, followed by time-dependent decreases. Establishing relationships between OF and plasma cannabinoid concentrations is essential for making inferences of impairment or other clinical outcomes from OF concentrations.     

Oral fluid cannabinoid concentrations following controlled smoked cannabis in chronic frequent and occasional smokers

Oral fluid (OF) is an alternative biological matrix for monitoring cannabis intake in drug testing, and drugged driving (DUID) programs, but OF cannabinoid test interpretation is challenging. Controlled cannabinoid administration studies provide a scientific database for interpreting cannabinoid OF tests. We compared differences in OF cannabinoid concentrations from 19 h before to 30 h after smoking a 6.8 % THC cigarette in chronic frequent and occasional cannabis smokers. OF was collected with the Statsure Saliva Sampler? OF device. 2D-GC-MS was used to quantify cannabinoids in 357 OF specimens; 65 had inadequate OF volume within 3 h after smoking. All OF specimens were THC-positive for up to 13.5 h after smoking, without significant differences between frequent and occasional smokers over 30 h. Cannabidiol (CBD) and cannabinol (CBN) had short median last detection times (2.5–4 h for CBD and 6–8 h for CBN) in both groups. THCCOOH was detected in 25 and 212 occasional and frequent smokers’ OF samples, respectively. THCCOOH provided longer detection windows than THC in all frequent smokers. As THCCOOH is not present in cannabis smoke, its presence in OF minimizes the potential for false positive results from passive environmental smoke exposure, and can identify oral THC ingestion, while OF THC cannot. THC?≥?1 μg/L, in addition to CBD?≥?1 μg/L or CBN?≥?1 μg/L suggested recent cannabis intake (≤13.5 h), important for DUID cases, whereas THC?≥?1 μg/L or THC?≥?2 μg/L cutoffs had longer detection windows (≥30 h), important for workplace testing. THCCOOH windows of detection for chronic, frequent cannabis smokers extended beyond 30 h, while they were shorter (0–24 h) for occasional cannabis smokers.     

Validation of a two-dimensional gas chromatography mass spectrometry method for the simultaneous quantification of cannabidiol, Δ9-tetrahydrocannabinol (THC), 11-hydroxy-THC, and 11-nor-9-carboxy-THC in plasma

A sensitive analytical method for simultaneous quantification of sub-nanogram concentrations of cannabidiol (CBD), Δ⁹-tetrahydrocannabinol (THC), 11-hydroxy-THC (11-OH-THC), and 11-nor-9-carboxy-THC (THCCOOH) in plasma is presented for monitoring cannabinoid pharmacotherapy and illicit cannabis use. Analytes were extracted from 1 mL plasma by solid-phase extraction, derivatized with N,O-bis(trimethylsilyl) trifluoroacetamide with 1% trimethylchlorosilane, and analyzed by two-dimensional gas chromatography mass spectrometry (2D-GCMS) with cryofocusing. The lower calibration curve was linear from 0.25–25 ng/mL for CBD and THC, 0.125-25 ng/mL for 11-OH-THC and 0.25-50 ng/mL for THCCOOH. A second higher linear range from 5–100 ng/mL, achieved through modification of injection parameters, was validated for THC, 11-OH-THC, and THCCOOH and was only implemented if concentrations exceeded the lower curve upper limit of linearity. This procedure prevented laborious re-extraction by allowing the same specimen to be re-injected for quantification on the high calibration curve. Intra- and inter-assay imprecision, determined at four quality control concentrations, were ≤7.8% CV. Analytical bias was within ±9.2% of target and extraction efficiencies were ≥72.9% for all analytes. Analytes were stable when stored at 22°C for 16 h, 4°C for 48 h, after three freeze–thaw cycles at −20°C and when stored on the autosampler for 48 h. This sensitive and specific 2D-GCMS assay provides a new means of simultaneously quantifying CBD, THC and metabolite biomarkers in clinical medicine, forensic toxicology, workplace drug testing, and driving under the influence of drugs programs.     

Studies on the metabolism of the Δ9‐tetrahydrocannabinol precursor Δ9‐tetrahydrocannabinolic acid A (Δ9‐THCA‐A) in rat using LC‐MS/MS,LC‐QTOF MS and GC‐MS techniques

In Cannabis sativa, Δ9‐Tetrahydrocannabinolic acid‐A (Δ9‐THCA‐A) is the non‐psychoactive precursor of Δ9‐tetrahydrocannabinol (Δ9‐THC). In fresh plant material, about 90% of the total Δ9‐THC is available as Δ9‐THCA‐A. When heated (smoked or baked), Δ9‐THCA‐A is only partially converted to Δ9‐THC and therefore, Δ9‐THCA‐A can be detected in serum and urine of cannabis consumers. The aim of the presented study was to identify the metabolites of Δ9‐THCA‐A and to examine particularly whether oral intake of Δ9‐THCA‐A leads to in vivo formation of Δ9‐THC in a rat model. After oral application of pure Δ9‐THCA‐A to rats (15 mg/kg body mass), urine samples were collected and metabolites were isolated and identified by liquid chromatography‐mass spectrometry (LC‐MS), liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) and high resolution LC‐MS using time of flight‐mass spectrometry (TOF‐MS) for accurate mass measurement. For detection of Δ9‐THC and its metabolites, urine extracts were analyzed by gas chromatography‐mass spectrometry (GC‐MS). The identified metabolites show that Δ9‐THCA‐A undergoes a hydroxylation in position 11 to 11‐hydroxy‐Δ9‐tetrahydrocannabinolic acid‐A (11‐OH‐Δ9‐THCA‐A), which is further oxidized via the intermediate aldehyde 11‐oxo‐Δ9‐THCA‐A to 11‐nor‐9‐carboxy‐Δ9‐tetrahydrocannabinolic acid‐A (Δ9‐THCA‐A‐COOH). Glucuronides of the parent compound and both main metabolites were identified in the rat urine as well. Furthermore, Δ9‐THCA‐A undergoes hydroxylation in position 8 to 8‐alpha‐ and 8‐beta‐hydroxy‐Δ9‐tetrahydrocannabinolic acid‐A, respectively, (8α‐Hydroxy‐Δ9‐THCA‐A and 8β‐Hydroxy‐Δ9‐THCA‐A, respectively) followed by dehydration. Both monohydroxylated metabolites were further oxidized to their bishydroxylated forms. Several glucuronidation conjugates of these metabolites were identified. In vivo conversion of Δ9‐THCA‐A to Δ9‐THC was not observed. Copyright © 2009 John Wiley & Sons, Ltd.     

Direct quantification of cannabinoids and cannabinoid glucuronides in whole blood by liquid chromatography–tandem mass spectrometry

The first method for quantifying cannabinoids and cannabinoid glucuronides in whole blood by liquid chromatography–tandem mass spectrometry (LC–MS/MS) was developed and validated. Solid-phase extraction followed protein precipitation with acetonitrile. High-performance liquid chromatography separation was achieved in 16 min via gradient elution. Electrospray ionization was utilized for cannabinoid detection; both positive (Δ⁹-tetrahydrocannabinol [THC] and cannabinol [CBN]) and negative (11-hydroxy-THC [11-OH-THC], 11-nor-9-carboxy-THC [THCCOOH], cannabidiol [CBD], THC-glucuronide, and THCCOOH-glucuronide) polarity were employed with multiple reaction monitoring. Calibration by linear regression analysis utilized deuterium-labeled internal standards and a 1/x ² weighting factor, yielding R ² values >0.997 for all analytes. Linearity ranged from 0.5 to 50 μg/L (THC-glucuronide), 1.0–100 μg/L (THC, 11-OH-THC, THCCOOH, CBD, and CBN), and 5.0–250 μg/L (THCCOOH-glucuronide). Imprecision was <10.5% CV, recovery was >50.5%, and bias within ±13.1% of target for all analytes at three concentrations across the linear range. No carryover and endogenous or exogenous interferences were observed. This new analytical method should be useful for quantifying cannabinoids in whole blood and further investigating cannabinoid glucuronides as markers of recent cannabis intake.     

Ethanol Production from H<Subscript>2</Subscript>SO<Subscript>3</Subscript>-Steam-Pretreated Fresh Sweet Sorghum Stem by Simultaneous Saccharification and Fermentation

The present work presents an alternative approach to ethanol production from sweet sorghum: without detoxification, acid-impregnated fresh sweet sorghum stem which contains soluble (glucose and sucrose) and insoluble carbohydrates (cellulose and hemicellulose) was steam pretreated under mild temperature of 100 °C. Simultaneous saccharification and fermentation experiments were performed on the pretreated slurries using Saccharomyces cerevisiae. Experimentally, ground fresh sweet sorghum stem was combined with H₂SO₃ at dosages of 0.25, 0.50, and 0.75 g/g dry matter (DM) and steam pretreated by varying the residence time (60, 120, or 240 min). According to enzymatic hydrolysis results and ethanol yields, H₂SO₃ was a powerful and mild acid for improving enzymatic digestibility of sorghum stem. At a solid loading of 10% (w/v) and acid dosage of 0.25 g/g DM H₂SO₃ at 100 °C for 120 min, 44.5 g/L ethanol was obtained after 48 ± 4 h of simultaneous saccharification and fermentation. This corresponded to an overall ethanol yield of 110% of the theoretical one, based on the soluble carbohydrates in the fresh sweet sorghum stem. The concentrations of hydroxymethylfurfural and furfural of the sulfurous acid pretreated samples were below 0.4 g/L. Ethanol would not inhibit the cellulase activity, at least under the concentration of 34 g/L.     

Direct Quantitation of Phytocannabinoids by One-Dimensional 1H qNMR and Two-Dimensional 1H-1H COSY qNMR in Complex Natural Mixtures

The widespread use of phytocannabinoids or cannabis extracts as ingredients in numerous types of products, in combination with the legal restrictions on THC content, has created a need for the development of new, rapid, and universal analytical methods for their quantitation that ideally could be applied without separation and standards. Based on previously described qNMR studies, we developed an expanded ¹H qNMR method and a novel 2D-COSY qNMR method for the rapid quantitation of ten major phytocannabinoids in cannabis plant extracts and cannabis-based products. The ¹H qNMR method was successfully developed for the quantitation of cannabidiol (CBD), cannabidiolic acid (CBDA), cannabinol (CBN), cannabichromene (CBC), cannabichromenic acid (CBCA), cannabigerol (CBG), cannabigerolic acid (CBGA), Δ9-tetrahydrocannabinol (Δ9-THC), Δ9-tetrahydrocannabinolic acid (Δ9-THCA), Δ8-tetrahydrocannabinol (Δ8-THC), cannabielsoin (CBE), and cannabidivarin (CBDV). Moreover, cannabidivarinic acid (CBDVA) and Δ9-tetrahydrocannabivarinic acid (Δ9-THCVA) can be distinguished from CBDA and Δ9-THCA respectively, while cannabigerovarin (CBGV) and Δ8-tetrahydrocannabivarin (Δ8-THCV) present the same ¹H-spectra as CBG and Δ8-THC, respectively. The COSY qNMR method was applied for the quantitation of CBD, CBDA, CBN, CBG/CBGA, and THC/THCA. The two methods were applied for the analysis of hemp plants; cannabis extracts; edible cannabis medium-chain triglycerides (MCT); and hemp seed oils and cosmetic products with cannabinoids. The ¹H-NMR method does not require the use of reference compounds, and it requires only a short time for analysis. However, complex extracts in ¹H-NMR may have a lot of signals, and quantitation with this method is often hampered by peak overlap, with 2D NMR providing a solution to this obstacle. The most important advantage of the COSY NMR quantitation method was the determination of the legality of cannabis plants, extracts, and edible oils based on their THC/THCA content, particularly in the cases of some samples for which the determination of THC/THCA content by ¹H qNMR was not feasible.     

Comparison of cannabinoid concentrations in oral fluid and whole blood between occasional and regular cannabis smokers prior to and after smoking a cannabis joint

A cross-over controlled administration study of smoked cannabis was carried out on occasional and heavy smokers. The participants smoked a joint (11 % Δ9-tetrahydrocannabinol (THC)) or a matching placebo on two different occasions. Whole blood (WB) and oral fluid (OF) samples were collected before and up to 3.5 h after smoking the joints. Pharmacokinetic analyses were obtained from these data. Questionnaires assessing the subjective effects were administered to the subjects during each session before and after the smoking time period. THC, 11-hydroxy-THC (11-OH-THC) and 11-nor-9-carboxy-THC (THCCOOH) were analyzed in the blood by gas chromatography or liquid chromatography (LC)-tandem mass spectrometry (MS/MS). The determination of THC, THCCOOH, cannabinol (CBN), and Δ9-tetrahydrocannabinolic acid A (THC-A) was carried out on OF only using LC-MS/MS. In line with the widely accepted assumption that cannabis smoking results in a strong contamination of the oral cavity, we found that THC, and also THC-A, shows a sharp, high concentration peak just after smoking, with a rapid decrease in these levels within 3 h. No obvious differences were found between both groups concerning THC median maximum concentrations measured either in blood or in OF; these levels were equal to 1,338 and 1,041 μg/L in OF and to 82 and 94 μg/L in WB for occasional and heavy smokers, respectively. The initial WB THCCOOH concentration was much higher in regular smokers than in occasional users. Compared with the occasional smokers, the sensation of confusion felt by the regular smokers was much less while the feeling of intoxication remained almost unchanged.

Ethanol Production from Residual Wood Chips of Cellulose Industry: Acid Pretreatment Investigation,Hemicellulosic Hydrolysate Fermentation,and Remaining Solid Fraction Fermentation by SSF Process

Current research indicates the ethanol fuel production from lignocellulosic materials, such as residual wood chips from the cellulose industry, as new emerging technology. This work aimed at evaluating the ethanol production from hemicellulose of eucalyptus chips by diluted acid pretreatment and the subsequent fermentation of the generated hydrolysate by a flocculating strain of Pichia stipitis. The remaining solid fraction generated after pretreatment was subjected to enzymatic hydrolysis, which was carried out simultaneously with glucose fermentation [saccharification and fermentation (SSF) process] using a strain of Saccharomyces cerevisiae. The acid pretreatment was evaluated using a central composite design for sulfuric acid concentration (1.0–4.0 v/v) and solid to liquid ratio (1:2–1:4, grams to milliliter) as independent variables. A maximum xylose concentration of 50 g/L was obtained in the hemicellulosic hydrolysate. The fermentation of hemicellulosic hydrolysate and the SSF process were performed in bioreactors and the final ethanol concentrations of 15.3 g/L and 28.7 g/L were obtained, respectively.     

Development of a liquid chromatography-mass spectrometry method for the determination of ursolic acid in rat plasma and tissue: application to the pharmacokinetic and tissue distribution study

A fast and sensitive liquid chromatography–mass spectrometry method was developed for the determination of ursolic acid (UA) in rat plasma and tissues. Glycyrrhetinic acid was used as the internal standard (IS). Chromatographic separation was performed on a 3.5 μm Zorbax SB-C18 column (30 mm × 2.1 mm) with a mobile phase consisting of methanol and aqueous 10 mM ammonium acetate using gradient elution. Quantification was performed by selected ion monitoring with (m/z)⁻ 455 for UA and (m/z)⁻ 469 for the IS. The method was validated in the concentration range of 2.5 − 1470 ng mL⁻¹ for plasma samples and 20 − 11760 ng g⁻¹ for tissue homogenates. The intra- and inter-day assay of precision in plasma and tissues ranged from 1.6% to 7.1% and 3.7% to 9.0%, respectively, and the intra- and inter-day assay accuracy was 84.2 − 106.9% and 82.1 − 108.1%, respectively. Recoveries in plasma and tissues ranged from 83.2% to 106.2%. The limits of detections were 0.5 ng mL⁻¹ or 4.0 ng g⁻¹. The recoveries for all samples were >90%, except for liver, which indicated that ursolic acid may metabolize in liver. The main pharmacokinetic parameters obtained were T _max = 0.42 ± 0.11 h, C _max = 1.10 ± 0.31 μg mL⁻¹, AUC = 1.45 ± 0.21 μg h mL⁻¹ and K _a = 5.64 ± 1.89 h⁻¹. The concentrations of UA in rat lung, spleen, liver, heart, and cerebellum were studied for the first time. This method is validated and could be applicable to the investigation of the pharmacokinetics and tissue distribution of UA in rats.     

Accurate quantification of mercapturic acids of styrene (PHEMAs) in human urine with direct sample injection using automated column-switching high-performance liquid chromatography coupled with tandem mass spectrometry

Styrene is one of the most important industrial chemicals, with an enormously high production volume worldwide. The urinary mercapturic acids of its metabolite styrene-7,8-oxide, namely N-acetyl-S-(2-hydroxy-1-phenylethyl)-l-cysteine (PHEMA 1) and N-acetyl-S-(2-hydroxy-2-phenylethyl)-l-cysteine (PHEMA 2), are specific biomarkers for the determination of individual internal exposure to this highly reactive intermediate of styrene. We have developed and validated a fast, specific and very sensitive method for the accurate determination of the sum of phenylhydroxyethyl mercapturic acids (PHEMAs) in human urine with an automated multidimensional liquid chromatography–tandem mass spectrometry method using ¹³C₆-labelled PHEMAs as internal standards. Analytes were stripped from the urinary matrix by online extraction on a restricted access material, transferred to the analytical column and subsequently determined by tandem mass spectrometry. The limit of quantification (LOQ) for the sum of PHEMAs was 0.3 μg/L urine and allowed us to quantify the background exposure of the (smoking) general population. Precision within series and between series ranged from 1.5 to 6.8% at three concentrations ranging from 3 to 30 μg/L urine; the mean accuracy was between 104 and 110%. We applied the method to spot urine samples from 40 subjects of the general population with no known occupational exposure to styrene. The median levels (range) for the sum of PHEMAs in urine of non-smokers (n = 22) were less than 0.3 μg/L (less than 0.3 to 1.1 μg/L), whereas in urine of smokers (n = 18), the median levels were 0.46 μg/L (less than 0.3 to 2.8 μg/L). Smokers showed a significantly higher excretion of the sum of PHEMAs (p = 0.02). Owing to its automation and high sensitivity, our method is well suited for application in occupational or environmental studies.     

The Correlations Between TCA-Glyoxalate Metabolite and Antibiotic Production of <Emphasis Type="Italic">Streptomyces</Emphasis> sp. M4018 Grown in Glycerol,Glucose, and Starch Mediums

The alterations of organic acids citrate, α-ketoglutarate, succinate, fumarate, malate production together with isocitrate lyase activity as a glyoxalate shunt enzyme, and antibiotic production of Streptomyces sp M4018 were investigated in relation to changes in the glucose, glycerol and starch concentrations (5–20 g/L) after identification as a strain of Streptomyces hiroshimensis based on phenotypic and genotypic characteristics. The highest intracellular citrate and α-ketoglutarate levels in 20 g/l of glucose, glycerol, and starch mediums were 399.47 ± 4.78, 426.93 ± 6.40, 355.84 ± 5.38 ppm and 444.81 ± 5.12, 192.96 ± 2.26, 115.20 ± 2.87 ppm, respectively. The highest succinate, malate, and fumarate levels were also determined in 20 g/l of glucose medium as 548.9 ± 11.21, 596.15 ± 8.26, and 406.42 ± 6.59 ppm and the levels were significantly higher than the levels in glycerol and starch. Extracellular organic acid levels measured also showed significant correlation with carbon source concentrations by showing negative correlation with pH levels of the growth medium. The antibiotic production of Streptomyces sp. M4018 was also higher in glucose medium as was the case also for organic acids when compared with glycerol. On the other hand, there is no production in starch.     

Simultaneous quantification of loureirin A and loureirin B in rat urine, feces, and bile by HPLC-MS/MS method and its application to excretion study

A simple HPLC-MS/MS method for simultaneous determination of loureirin A and loureirin B in rat urine, feces, and bile after oral administration of 10.6 g/kg of longxuejie (one rare traditional Chinese medicinal herb) was developed for the first time. The analytes and buspirone (internal standard) were separated on a C₅ column with acetonitrile–water (containing 0.1% formic acid) as mobile phase at a flow rate of 0.4 min/mL. The detector was a Q-trap™ mass spectrometer with an electrospray ionization interface operating in the multiple reaction monitoring mode. Calibration curves of loureirin A in rat urine, feces, and bile were linear over the concentration range of 1.00–5,000 ng/mL. Loureirin B in rat urine, feces, and bile ranged between 0.08 and 20, 0.20 and 20, and 0.10 and 500 ng/mL, respectively. Validation revealed that the method was specific, accurate, and precise. The fully validated method was applied to the excretion study of loureirin A and loureirin B in rats. After oral administration of 10.6 g/kg longxuejie, cumulative excretion amount of loureirin A and loureirin B in rat urine were 2.94 ± 0.81 and 0.36 ± 0.16 μg at 72 h, respectively. Of the total dose, 5.35% of loureirin A and 5.46% of loureirin B were excreted from feces at 60 h. The cumulative amounts of loureirin A and loureirin B in rat bile reached 4.49 ± 0.98 and 5.11 ± 0.83 μg, respectively, at 36 h after dosing, accounting for 0.054% and 0.056% of the total dose.     

A Process to Produce Penicillin G Acylase by Surface-Adhesion Fermentation Using <Emphasis Type="Italic">Mucor griseocyanus</Emphasis> to Obtain 6-Aminopenicillanic Acid by Penicillin G Hydrolysis

The production of extracellular and mycelia-associated penicillin G acylase (maPGA) with Mucor griseocyanus H/55.1.1 by surface-adhesion fermentation using Opuntia imbricata, a cactus, as a natural immobilization support was studied. Enzyme activity to form 6-aminopencillanic acid (6-APA) from penicillin G was assayed spectrophotometrically. The penicillin G hydrolysis to 6-APA was evaluated at six different times using PGA samples recovered from the skim milk medium at five different incubation times. Additionally, the effect of varying the penicillin G substrate concentration level on the PGA enzyme activity was also studied. The maximum reaction rate, V _max, and the Michaelis constant, K _M, were determined using the Michaelis–Menten model. The maximum levels for maPGA and extracellular activity were found to be 2,126.50 international unit per liter (IU/l; equal to 997.83 IU/g of support) at 48 h and 755.33 IU/l at 60 h, respectively. Kinetics of biomass production for total biomass showed a maximum growth at 60 h of 3.36 and 2.55 g/l (equal to 0.012 g of biomass per gram of support) for the immobilized M. griseocyanus biomass. The maPGA was employed for the hydrolysis of penicillin G to obtain 6-APA in a batch reactor. The highest quantity of 6-APA obtained was 226.16 mg/l after 40-min reaction. The effect of substrate concentration on maPGA activity was evaluated at different concentrations of penicillin G (0–10 mM). K _M and V _max were determined to be 3.0 × 10⁻³ M and 4.4 × 10⁻³ mM/min, respectively.     

Determination of cyanide in blood by electrospray ionization tandem mass spectrometry after direct injection of dicyanogold

An electrospray ionization tandem mass spectrometric (ESI-MS-MS) method has been developed for the determination of cyanide (CN^–) in blood. Five microliters of blood was hemolyzed with 50 μL of water, then 5 μL of 1 M tetramethylammonium hydroxide solution was added to raise the pH of the hemolysate and to liberate CN^– from methemoglobin. CN^– was then reacted with NaAuCl₄ to produce dicyanogold, Au(CN)₂^–, that was extracted with 75 μL of methyl isobutyl ketone. Ten microliters of the extract was injected directly into an ESI-MS-MS instrument and quantification of CN^– was performed by selected reaction monitoring of the product ion CN^– at m/z 26, derived from the precursor ion Au(CN)₂^– at m/z 249. CN^– could be measured in the quantification range of 2.60 to 260 μg/L with the limit of detection at 0.56 μg/L in blood. This method was applied to the analysis of clinical samples and the concentrations of CN^– in the blood were as follows: 7.13 ± 2.41 μg/L for six healthy non-smokers, 3.08 ± 1.12 μg/L for six CO gas victims, 730 ± 867 μg for 21 house fire victims, and 3,030 ± 97 μg/L for a victim who ingested NaCN. The increase of CN^– in the blood of a victim who ingested NaN₃ was confirmed using MS-MS for the first time, and the concentrations of CN^– in the blood, gastric content and urine were 78.5 ± 5.5, 11.8 ± 0.5, and 11.4 ± 0.8 μg/L, respectively.     

Detection of Cry1Ab toxin in the leaves of MON 810 transgenic maize

The distribution of Cry1Ab toxin was detected in the leaves of genetically modified maize of genetic event MON 810 by enzyme-linked immunosorbent assay. Cry1Ab toxin contents in the leaves at reproductive (milk, R3) phenological stage were measured to be between 3,878 and 11,148 ng Cry1Ab toxin/g fresh weight. Toxin content was significantly lesser (significant difference (SD) = 1,823 ng Cry1Ab toxin/g fresh leaf weight, p < 0.01) in leaves at the lowest leaf level, than at higher leaf levels, probably due to partial leaf necrotisation. A substantial (up to 22%) plant-to-plant variation in Cry1Ab contents in leaves was observed. When studying toxin distribution within the cross and longitudinal sections of single leaves, lesser variability was detected diagonally, with approximately 20% higher toxin concentrations at or near the leaf vein. More significant variability (SD = 2,220 ng Cry1Ab toxin/g fresh leaf weight, p < 0.01) was seen lengthwise along the leaf, starting at 1,892 ng Cry1Ab toxin/g fresh weight at the sheath and rising to maximum concentration at the middle of the lamella. Cry1Ab toxin content may suffer significant (SD = 2,230 ng Cry1Ab toxin/g fresh leaf weight, p < 0.01) decreases in the leaf due to necrotisation. The results indicate that the longitudinal dimension of the leaf has more significance for sampling purposes than the diagonal position.     

An analytical approach for on-site analysis of breath samples for Δ9-tetrahydrocannabinol (THC)

Increased acceptance of cannabis containing the psychoactive component, Δ9-tetrahydrocannabinol (THC), raises concerns about the potential for impaired drivers and increased highway accidents. In contrast to the “breathalyzer” test, which is generally accepted for determining the alcohol level in a driver, there is no currently accepted roadside test for THC in a motorist. There is a need for an easily collectible biological sample from a potentially impaired driver coupled with an accurate on-site test to measure the presence and quantity of THC in a driver. A novel breath collection device is described, which includes three separate sample collectors for collecting identical A, B, and C breath samples from a subject. A simple one-step ethanol extraction of the “A” breath collector sample can be analyzed by UHPLC/selected ion monitoring (SIM) liquid chromatography/mass spectrometry (LC/MS) to provide qualitative and quantitative determination of THC in breath sample in less than 4 min for samples collected up to 6 h after smoking a cannabis cigarette. SIM LC/MS bioanalyses employed d3-THC as the stable isotope internal standard fortified in negative control breath samples for quantitation including replicates of six calibrator standards and three quality control (QC) samples. Subsequent confirmation of the same breath sample in the B collectors was then confirmed by a reference lab by LC/MS/MS analysis. Fit-for-purpose bioanalytical validation consistent with pharmaceutical regulated bioanalyses produced pharmacokinetic (PK) curves for the two volunteer cannabis smokers. These results produced PK curves, which showed a rapid increase of THC in the breath of the subjects in the first hour followed by reduced THC levels in the later time points. A simpler single-point calibration curve procedure with calibrators and QC prepared in ethanol provided similar results. Limitations to this approach include the higher cost and operator skill sets for the instrumentation employed and the inability to actually determine driver impairment.     

Continuous Production of Ethanol from Starch Using Glucoamylase and Yeast Co-Immobilized in Pectin Gel

This work presents a continuous simultaneous saccharification and fermentation (SSF) process to produce ethanol from starch using glucoamylase and Saccharomyces cerevisiae co-immobilized in pectin gel. The enzyme was immobilized on macroporous silica, after silanization and activation of the support with glutaraldehyde. The silica–enzyme derivative was co-immobilized with yeast in pectin gel. This biocatalyst was used to produce ethanol from liquefied manioc root flour syrup, in three fixed bed reactors. The initial reactor yeast load was 0.05 g wet yeast/ml of reactor (0.1 g wet yeast/g gel), used in all SSF experiments. The enzyme concentration in the reactor was defined by running SSF batch assays, using different amount of silica–enzyme derivative, co-immobilized with yeast in pectin gel. The chosen reactor enzyme concentration, 3.77 U/ml, allowed fermentation to be the rate-limiting step in the batch experiment. In this condition, using initial substrate concentration of 166.0 g/l of total reducing sugars (TRS), 1 ml gel/1 ml of medium, ethanol productivity of 8.3 g/l/h was achieved, for total conversion of starch to ethanol and 91% of the theoretical yield. In the continuous runs, feeding 163.0 g/l of TRS and using the same enzyme and yeast concentrations used in the batch run, ethanol productivity was 5.9 g ethanol/l/h, with 97% of substrate conversion and 81% of the ethanol theoretical yield. Diffusion effects in the extra-biocatalyst film seemed to be reduced when operating at superficial velocities above 3.7 × 10⁻⁴ cm/s.