Altered morphine glucuronide and bile acid disposition in patients with nonalcoholic steatohepatitis

The functional impact of altered drug transport protein expression on the systemic pharmacokinetics of morphine, hepatically-derived morphine glucuronide (morphine-3- and morphine-6-glucuronide), and fasting bile acids was evaluated in patients with biopsy-confirmed non-alcoholic steatohepatitis (NASH) compared to healthy subjects. The maximum concentration (Cmax) and area under the concentration-time curve (AUC0-last) of morphine glucuronide in serum were increased in NASH patients (343 vs. 225nM and 58.8 vs. 37.2μM*min, respectively; P≤0.005); morphine pharmacokinetics did not differ between groups. Linear regression analyses detected an association of NASH severity with increased morphine glucuronide Cmax and AUC0-last (P<0.001). Fasting serum glycocholate, taurocholate and total bile acid concentrations were associated with NASH severity (P<0.006). Increased hepatic basolateral efflux of morphine glucuronide and bile acids is consistent with altered hepatic transport protein expression in patients with NASH and may partially explain differences in efficacy and/or toxicity of some highly transported anionic drugs/metabolites in this patient population

A Gas-Liquid Chromatographic Method for Quantitation of 1,3-Butylene Glycol in Whole Blood or Plasma and the Separation of the Short Chain Glycols

A microanalytical method with direct on-column specimen injection for determination of 1,3-butylene glycol (1,3-butanediol) in whole blood or plasma using gas-liquid chromatography with flame ionization is described. Whole blood or serum (minimum of 10 μL) was mixed with an equal volume of internal standard (1,2-propanediol, 50 mg/dL) and a 2-μL aliquot was injected onto the column without prior derivatization or extraction. The other short chain (C2 to C4) alkyldiols were separated by this method and did not interfere with the quantitation of 1,3-butylene glycol. The method was linear (y = 0.0206x + [-0.0073], r = 0.9990) over the range of 25 to 100 mg/dL and the coefficient of variation varied between 0.74 and 6.03%. Minimum detectable concentration of 1,3-butylene glycol was 5.0 mg/dL. The method described is suitable for the rapid detection of potentially toxic blood or plasma levels of 1,3-butylene glycol, as well as for the detection of other short chain glycols

Application of Micellar Electrokinetic Capillary Chromatography to Forensic Analysis of Barbiturates in Biological Fluids

Micellar electrokinetic capillary chromatography (MECC) is a form of capillary zone electrophoresis. Addition of a surfactant produces micelles in an aqueous/organic buffer. Separation of drugs is obtained via differences in the electrophoretic mobilities of the analytes within the capillary, resulting from their electrophoretic velocity and the electroosmotic flow of the buffer in a given electric field. The migration order is determined by the differential partitioning of the drugs between the micelles and the aqueous/organic phase. Barbiturates were extracted from various biological fluids at pH 4.5 with TOXI-TUBES B. MECC analyses were performed using a Waters Quanta 4000 Capillary Electrophoretic System with a 745 Data Module with a 75 μ x 60 cm capillary and an aqueous/organic buffer of 85% 10 mM borate, 10 mM phosphate, 100 mM sodium dodecyl sulfate and 15% acetonitrile at a pH of 8.5 with a voltage of 20 kV using ultraviolet absorption detection at 214 nm. Migration times were: phenobarbital, 7.78 min.; butalbital, 8.01 min.; butabarbital, 8.23 min.; mephobarbital (internal standard), 8.88 min.; amobarbital, 9.41 min.; pentobarbital, 10.03 min. and secobarbital, 10.79 min. Correlation coefficients (r) between peak areas and concentration ranges of 3 to 60 μg/mL were from 0.964 to 0.999. Coefficients of variation (CV) raged from 2.6 to 8.6% between days and 2.3 to 9.8% within day. Application of this methodology to four forensic cases of butalbital intoxication detected concentrations of 0.7 to 12.7 μg/mL in blood; 0.8 to 1.9 μg/mL in vitreous humor and 1.5 to 7.6 μg/mL in urine. MECC is applicable to forensic analysis of barbiturates extracted from biological fluids

Postmortem Determination of the Biological Distribution of Sufentanil and Midazolam After an Acute Intoxication

A case is presented of a death caused by self-injection of sufentanil and midazolam. Biological fluids and tissues were analyzed for midazolam by high performance liquid chromatography (HPLC) and gas chromatography/mass spectrometry (GC/MS) and for sufentanil by GC/MS. Midazolam was extracted from basified fluids or tissues homogenated with n-butyl chloride and analyzed by HPLC by using a phosphate buffer: acetonitrile (60:40) mobile phase on a μ-Bondapak C18 column at 240 nm. Sufentanil was extracted from basified fluids and tissue homogenates with hexane:ethanol (19:1). GC/MS methodology for both compounds consisted of chromatographic separation on a 15-m by 0.25-mm inside diameter (ID) DB-5 (1.0-μm-thick film) bonded phase fused silica capillary column with helium carrier (29 cm/s) splitless injection at 260°C; column 200°C (0.8 min) 10°C/min to 270°C; and electron ionization and multiple ion detection for midazolam (m/z 310), methaqualone (IS, m/z 235), sufentanil (m/z 289), and fentanyl (IS, m/z 245). Sufentanil concentrations were: blood 1.1 ng/mL, urine 1.3 ng/mL, vitreous humor 1.2 ng/mL, liver 1.75 ng/g, and kidney 5.5 ng/g. Midazolam concentrations were: blood 50 ng/mL, urine 300 ng/mL, liver 930 ng/g, and kidney 290 ng/g. Cause of death was attributed to an acute sufentanil/midazolam intoxication and manner of death a suicide

Spectral Differentiation and Gas Chromatographic/Mass Spectrometric Analysis of the Lacrimators 2-Chloroacetophenone and O-Chlorobenzylidene Malononitrile

2-Chloroacetophenone (CN) and o-chlorobenzylidene malononitrile (CS) are the most common chemical agents used as lacrimators in the United States. There is lack of complete spectral data on these compounds in the literature. Spectral data (ultraviolet, fluorescence, proton nuclear magnetic resonance, and infrared), and a gas-liquid chromatographic/mass spectrometric method are presented that differentiate and identify CN and CS. These methods and data were used to identify a forensic science specimen from an accidental intoxication

A Fatal Interaction of Methocarbamol and Ethanol in an Accidental Poisoning

A case is presented of a fatal drug interaction caused by ingestion of methocarbamol (Robaxin®) and ethanol. Methocarbamol is a carbamate derivative used as a muscle relaxant with sedative effects. Therapeutic concentrations of methocarbamol are reported to be 24 to 41 μ/mL. Biological fluids were screened for ethanol using the Abbott TDx system and quantitated by gas-liquid chromatography (GLC). Determination of methocarbamol concentrations in biological tissue homogenates and fluids were obtained by colorimetric analysis of diazotized methocarbamol. Blood ethanol concentration was 135 mg/dL (0.135% w/v) and urine ethanol was 249 mg/dL (0.249% w/v). Methocarbamol concentrations were: blood, 257 μg/mL; bile, 927 μg/mL; urine, 255 μg/mL; gastric, 3,7 g; liver, 459 μg/g; and kidney, 83 μg/g. The combination of ethanol and carbamates is contraindicated since acute alcohol intoxication combined with carbamate usage can lead to combined central nervous system depression as a result of the interactive sedative-hypnotic properties of the compounds

Capillary Ion Electrophoresis of Endogenous Anions and Anionic Adulterants in Human Urine

Normal human urine contains many anions and cations. Ionic concentrations in urine have classically been determined by spectrophotometry of color reactions, flame emission spectrophotometry, atomic absorption spectrophotometry, high performance liquid chromatography, or potentiometry with ion-specific electrodes. Capillary ion electrophoresis (CIE) is a form of capillary electrophoresis which uses the differential electrophoretic mobility of ions to perform a separation of an ionic mixture. Various salts can be added to urine specimens to abnormally elevate ionic concentrations and interfere with either immunoassay urine drug screening procedures or gas chromatographic/mass spectrometric confirmation techniques. Application of CIE for the direct detection of endogenous anions and anionic adulterants in human urine specimens was the purpose of this investigation. CIE was performed using a Waters Quanta 4000 Capillary Electrophoresis System with either direct or indirect ultraviolet absorption detection at 254 nm. CIE of 30 random normal urine specimens and 21 urine specimens suspected of adulteration was performed. Duplicate aliquots were assayed by CIE and by colorimetric technique for nitrite. Sixteen specimens had elevated concentrations of nitrite and/or nitrate. The correlation coefficient between nitrite CIE and colorimetric results was 0.9895. Three specimens had detectable concentrations of chromate and were suspected of being adulterated with Urine Luck, an adulterant found to contain chromate. Two specimens suspected of being adulterated with bleach were found to only contain chloride, sulfate, and phosphate. CIE is applicable to forensic analysis of urine anion concentrations. CIE can easily quantitate numerous endogenous anions and offers a method to detect and/or confirm anion adulteration of urine specimens

Poisoning From Oral Ingestion of Carbofuran (Furadan 4F), a Cholinesterase-Inhibiting Carbamate Insecticide, and Its Effects on Cholinesterase Activity in Various Biological Fluids

A case is presented of a fatal ingestion of Furadan (carbofuran), a cholinesterase-inhibiting carbamate insecticide. A 26-year-old white male was found dead with a partially filled 1-gal (3.8-L) container of Furadan 4F insecticide-nematocide (44.9% carbofuran). The individual had ingested approximately 345 mL of the mixture. Analysis of cholinesterase activity in various biological fluids was performed spectrophotometrically using propionylthiocholine and 5,5\u27-dithiobis-2-nitrobenzoic acid [Sigma Diagnostics, cholinesterase procedure No. 422 (PTC)] which was measured at 405 nm and 30°C in a Gilford Stasar III Spectrophotometer. The cholinesterase activities were as follows: plasma, 245 units (U)/L (93% inhibition/7% normal activity); serum, 208 U/L (95.3% inhibition/4.7% normal activity); whole blood, 297 U/L (92.8% inhibition/7.2% normal activity); erythrocytes, 58 U/L (99% inhibition/1% normal activity); vitreous humor, 7 U/L; and bile, 148 U/L. Carbofuran was detected in the blood and gastric contents by thin-layer chromatography. No alcohol or other drugs were detected in the blood, urine, or gastric contents. Ingestion of the carbofuran produced acute visceral congestion and pulmonary edema. Death was caused by anoxia due to respiratory paralysis produced by cholinesterase inhibition from Furadan (carbofuran) ingestion

GC/MS Method for the Determination of Flumazenil in Plasma and Urine

Flumazenil (FMZ, Flumazepil, Anexate, RO 15-1788) is an imidiazobenzodiazepine used in the reversal of benzodiazepine sedation and intoxication. Dosages of up to 1 mg reverse the coma and respiratory and CNS depression produced by benzodiazepines. Therapeutic dosages of FMZ produce plasma concentrations of up to 500 ng/mL. An analytical method was developed to determine FMZ concentrations in plasma and urine. Plasma and urine samples were spiked with FMZ from 5 to 500 ng/mL and flunitrazepam (FN, internal standard, 500 ng/ml). Specimens (3 ml) were extracted using 9 ml n-butyl chloride and 3 ml ammonium chloride buffer (pH 9.2). Extracts were dried under N2 in silanized glass, reconstituted in 25 μl methanol and 5 μl injected. GC/MS methodology utilized 15 m x 0.25 mm (ID) DB-5 (1.0 μm film) fused silica capillary column, with He carrier (29 cm/sec), splitless injection at 270°C, column 200-270°C @ 10°/min, separator oven 270°C, electron ionization (70 eV) with selective ion monitoring for FMZ (303 m/z) and FN (312 m/z). FMZ eluted at 7.37 min and FN at 8.19 min (relative index 0.8998). Correlation coefficients of concentration to peak area ratios were r = 0.9979 for plasma and r = 0.9943 for urine. Coefficients of variation were: plasma, 25 ng/ml, 13.2% (n = 5, x = 23 ± 1.3); plasma, 250 ng/ml, 6.6% (n = 6, x = 274 ± 7.4); urine, 25 ng/ml, 12.1% (n = 5, x = 23 ± 1.2); urine, 250 ng/ml, 10.7% (n = 5, x = 308 ± 14.7). This GC/MS method can determine FMZ concentrations in plasma and urine associated with therapeutic doses of FMZ

Iontophoresis of Dexamethasone-Phosphate Into the Equine Tibiotarsal Joint

In human rehabilitation medicine, dexamethasone-phosphate is theoretically iontophoresed to localized subcutaneous tissue where conversion to dexamethasone occurs. This delivery system has recently been introduced into veterinary medicine for the same purpose. However, the pharmacokinetic justification for parenteral delivery of this prodrug remains undocumented. Utilizing iontophoretic methods that are relevant to both human and veterinary clinical practice, the present investigation compared injection and iontophoresis of dexamethasone-phosphate into the equine tibiotarsal joint, also known as the tarsocrual joint. The tibiotarsal joints of seven horses were injected with 4 mL of 6 mg/mL dexamethasone-phosphate. With a similar drug concentration and over the same application site, six different horses underwent simultaneous cathodic iontophoresis (4 mA, 40 min) or passive application (0 mA, 40 min) on contralateral limbs. Following all applications, tibiotarsal joint synovium was collected. Local venous blood samples were also collected from the iontophoretic and passive application sites for analysis of plasma drug concentrations. Because of the potential for conversion of dexamethasone-phosphate to dexamethasone, an extraction and analysis protocol was developed for both chemicals. The technique demonstrated a linear range of detection (0.39-12 μg/mL) and a capability for measuring both chemicals in plasma and synovium. Conversion of dexamethasone-phosphate to dexamethasone occurred during synovial incubation (37 °C) and following freeze-thaw cycles. In contrast to the measurable synovial concentrations of dexamethasone-phosphate (2.3±0.96 mg/mL) and dexamethasone (0.27±0.07 mg/mL) following injection, neither drug was detected in the synovium or the local venous blood following iontophoretic or passive applications. In conclusion, these results do not confirm iontophoretic or passive delivery of measurable dexamethasone-phosphate into the tibiotarsal joint using current clinical methods