High-performance Liquid Chromatography-electrospray Mass Spectrometry Research Articles

Preparation of low molecular weight chondroitin sulfate from different sources by H2O2/ascorbic acid degradation and its degradation mechanism

Low molecular weight chondroitin sulfate (LMCS) has attention for enhanced bioavailability and bioactivity compared to native CS. We optimized H2O2/ ascorbic acid (Vc) degradation conditions to prepare LMCS from chicken, bovine, and shark cartilages. Degradation kinetics models and chemical composition data of LMCS showed the GlcA residues of chondroitin-4-sulfate (CSA) may be preferentially attacked. Nuclear magnetic resonance (NMR) spectroscopy and high-performance liquid chromatography-electrospray mass spectrometry (HPLC-MS) indicated that the CH of GlcA in CS was broken through a hydrogen abstraction reaction to break the β-(1 → 3) bond and form the hexendioic acid product. Standard density functional theory (DFT) calculations indicated that the energy required for the hydrogen abstraction from the C1-H bond in GlcA was lower than that of GalNAc. Molecular dynamics (MD) showed that CSA was more likely to interact with hydroxyl radicals (·OH) than non-sulfated chondroitin (CSO) and chondroitin-6-sulfate (CSC). These results provide guidance for producing LMCS.

Coelomic fluid of Echinometra mathaei: The new prospects for medicinal antioxidants

Echinoid pigments have various biological properties such as antioxidant, cytotoxic, and antibacterial activities. We aimed to evaluate the extraction of cell-free coelomic fluid (CFCF) and coelomocyte lysate (CL) as well as qualitatively and quantitatively identify the coelomic fluid of Echinometra mathaei as a new source of polyhydroxylatednaphthoquinone (PHNQ) antioxidant pigments. Based on the High Performance liquid chromatography–electrospray mass spectrometry (HPLC-MS) analysis in negative mode, the main quinonoid (PHNQ) pigments were identified and quantified. This study also illustrated the total ion current chromatograms and related mass spectra of Spinochrome A, Spinochrome B, Spinochrome C, and Echinochrome A in CL and SpinochromeC in CFCF samples. The ions at 221, 279, 265 and 263 m/z correspond to the pseudo-molecular [M − H] ions of Spinochrome B, Spinochrome C, Echinochrome A, and Spinochrome A, respectively. These components have previously been noted from the shells and spines of sea urchins but identification of PHNQs pigments in CL and CFCF of E. mathaei using LC-MS was introduced for the first time. The results also showed that, the highest DPPH radical scavenging activity of CFCF (88.12 DPPH% scavenging at 70 μg/mL, IC50 = <10 μg/mL). The findings clearly suggest that the coelomic fluid of E. mathaei could be served as the promising as well as potential natural antioxidants in the medical and pharmaceutical industries and could replace the increasing prices of the commercial antioxidants products.

Antioxidant and Antidiabetic Activity of Proanthocyanidins from Fagopyrum dibotrys.

Proanthocyanidins are natural glycosidase inhibitors with excellent antioxidant activity. This study aims to search for a new source of proanthocyanidins for the prevention and treatment of type 2 diabetes with higher content and better activity and get their structure elucidated. First, the total proanthocyanidins contents (TOPCs), antioxidant activity, antidiabetic activity of seven common Polygonaceae plants were analyzed and compared. Then proanthocyanidins from the rhizome of Fagopyrum dibotrys were purified, and the detailed structure was comprehensively analyzed by ultraviolet visible spectroscopy (UV-Vis), Fourier transform infrared spectroscopy (FT-IR), 13C nuclear magnetic resonance spectroscopy (13C NMR), reversed-phase high-performance liquid chromatography-electrospray mass spectrometry (RP-HPLC-ESI-MS), and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). The rhizome of F. dibotrys showed the highest TOPCs, the strongest antioxidant, and antidiabetic activities; the TOPCs, antioxidant and antidiabetic activities were all very significantly positively correlated. Proanthocyanidins purified from the rhizome of F. dibotrys showed better antidiabetic activity than grape seed proanthocyanidins (GsPs). Seventy-two proanthocyanidins from trimer to undecamer with a mean degree of polymerization (mDP) of about 5.02 ± 0.21 were identified with catechin and epicatechin as the dominant monomers. Conclusion: Proanthocyanidins are the main antioxidant and antidiabetic active substances of F. dibotrys and are expected to be developed into potential antioxidant and hypoglycemic products.

Phlorotannins Composition, Radical Scavenging Capacity and Reducing Power of Phenolics from the Brown Alga Cystoseira sauvageauana

ABSTRACT The aim of this study was to assess the phenolic composition extracted from the brown alga Cystoseira sauvageauana collected from the Algerian coast through high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) analysis and their antioxidant activity based on free radical scavenging and reducing power. The crude extract was obtained from dried biomass using ethanol/water and then fractionated (liquid-liquid) using solvents of increasing polarity. The ethyl acetate fraction contained the largest amount of phenolic compounds (59.27 ± 0.20 g of phloroglucinol equivalents per 100 g of fraction dry weight) and showed strong radical scavenging and reducing capacity. HPLC-ESI-MS/MS analysis using the multiple reactions monitoring scan mode showed that this fraction was mostly composed of phlorotannins (41 compounds) with a degree of polymerization ranging from 3 to 12. The most abundant compounds were two phloroglucinol nonamer isomers (deprotonated molecular ion [M-H]− at m/z 1117). The results suggest that Cystoseira sauvageauana represents an important natural source of antioxidants that could be valorized for human health.

Pyrrolizidine alkaloids in floral honeys of tropical Ghana: a health risk assessment

ABSTRACTThere is a vast amount of information about the nutritional and medicinal properties of honey as a result of its numerous benefits. However, honeys have been found to be contaminated with hepatotoxic and carcinogenic pyrrolizidine alkaloids (PAs) on account of bees foraging on PA-containing plants. This study deals with the analysis of PAs in tropical honeys emanating from different agro-ecological zones of Ghana in order to assess its potential health risk. PAs of 48 honey samples were analysed using high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). The results show that a total of 85% of the honeys from various agro-ecological zones were PA positive including all honeys from supermarkets. The highest concentration of PAs was 2639 μg kg−1, while the average PA concentration of the samples was 283 μg kg−1. The study also found Chromolaena odorata pollens in majority of the honeys, thus indicating the plant as major source of PA contamination of honeys in the tropical regions.

Photodegradation of dimethenamid-P in deionised and ground water

The study of photodegradation of dimethenamid-P herbicide was performed in deionised and ground water using TiO2 as a catalyst under UV light. The effect of electron acceptor (H2O2), scavenger of ?OH radicals (C2H5OH) and scavenger of holes (NaCl and Na2SO4) as well as solution pH was analyzed. The photodegradation of dimethenamid-P was followed by HPLC. The formation of transformation products was followed using high performance liquid chromatography-electrospray mass spectrometry. Ion chromatography and total organic carbon measurements were used for the determination of the mineralization level. HPLC analysis showed the almost complete removal of herbicide after 90 min in deionised and ground water, while total organic carbon analysis showed that dimethenamid-P was mineralized 64 and 50 % in deionised and ground water, respectively. The ion chromatography results showed that the mineralization process leads to the formation of chloride, sulphate and nitrate anions during the process. Transformation products were identified and the degradation mechanism was proposed.

A sensitive LC–ESI-MS/MS method for the quantification of avobenzone in rat plasma and skin layers: Application to a topical administration study

This study describes the development of a sensitive high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method for the quantification of avobenzone in rat plasma and skin layers. Separations were performed on a Zorbax SB C8 column using a binary gradient mobile phase composed of acetonitrile and 0.1% formic acid in water. The assay achieved LLOQ of 0.5ng/ml for plasma, 5ng/ml for stratum corneum, and 10ng/ml for epidermis and dermis. This method was applied to a percutaneous absorption study of avobenzone in rats. At 12h following topical application of emulsion and lotion (applied amount of avobenzone 11.7mg/kg), avobenzone was found primarily in the stratum corneum (16.3-17.8%) followed by epidermis (2.0-3.4%) and dermis (0.11-0.15%). Avobenzone was not quantifiable in the plasma samples collected over a 12h sampling period. Given the excellent plasma assay sensitivity, this study provides evidence that the systemic absorption of avobenzone is insignificant, if any, after topical application.

High-Performance Liquid Chromatography–Electrospray Ionization Tandem Mass Spectrometry for Metabolism Study of Timosaponin AIII

A sensitive method based on high-performance liquid chromatography-electrospray ionization tandem mass spectrometry was developed for the determination of timosaponin AIII (TA3) and its in vivo and in vitro metabolites. The rat plasma, urine, feces and tissue samples were collected after oral administration of TA3 at a single dose of 300 mg/kg. TA3 was incubated into artificial gastric juice and artificial intestinal juice. The in vivo and in vitro samples were purified by using liquid-liquid extraction. The structures of metabolites were elucidated by comparing their molecular weights, retention times and tandem mass spectrometric spectra with those of the parent drug. As a result, four metabolites (deglycosylated TA3, two hydroxylated TA3 and timosaponin BII) and the parent drug were found in in vivo and in vitro samples. In addition to the parent drug, one, one and two metabolites were identified in heart, urine and feces, respectively. Only the parent drug was detected in plasma, liver and kidney. One hydroxylation metabolite and TA3 were identified from incubation samples with AGJ, whereas two hydroxylation metabolites and TA3 were detected from the incubation with AIJ. This is the first systematic metabolism study of TA3. The biotransformation pathways of TA3 primarily included deglycosylation, hydroxylation and glycosylation.

SIMULTANEOUS DETERMINATION OF FOUR FLAVONOID AGLYCONE ANALOGS IN THE INFLORESCENCE OF JUGLANS REGIA L. BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY-DIODE ARRAY DETECTION

A reversed-phase high-performance liquid chromatography coupled with a diode array detection method and a high-performance liquid chromatography-electrospray ionization tandem mass spectrometry method were first developed and validated for the simultaneous determination of four flavonoid aglycone analogs in the inflorescence of Juglans regia L. grown in different areas of Gansu province, China, surmounting the challenges rising from the complexity of the matrices owing to different origins of the plant. The separation of the four compounds was accomplished through a ternary mobile phase, with good specificity and satisfactory resolution. The chromatographic peaks of the four investigated compounds were identified by comparing their retention time and mass spectrometric profile with the corresponding reference compounds. The method showed a good linearity (r2 > 0.9998), high precision (RSD <5%), and recoveries (ranged from 96.6–102.7%) were acceptable. The limits of detection (LOD) and the limits of quantification (LOQ) are ranged from 0.26–0.58 µg/ml and from 0.86–1.90 µg/ml, respectively. The results demonstrated that this analytical approach is ideal for the determination of such bioactive compounds as flavonoid aglycones, and that it constructed a basis for the comprehensive evaluation of the quality of Juglans regia L.

Using a Build-and-Click Approach for Producing Structural and Functional Diversity in DNA-Targeted Hybrid Anticancer Agents

An efficient screening method was developed for functionalized DNA-targeted platinum-containing hybrid anticancer agents based on metal-mediated amine-to-nitrile addition, a form of "click" chemistry. The goal of the study was to generate platinum-acridine agents for their use as cytotoxic "warheads" in targeted and multifunctional therapies. This was achieved by introducing hydroxyl, carboxylic acid, and azide functionalities in the acridine linker moiety and by varying the nonleaving groups attached to platinum. The assay, which was based on microscale reactions between 6 platinum-nitrile complexes and 10 acridine derivatives, yielded a small library of 60 platinum-acridines. Reactions were monitored, and product mixtures were quantitatively analyzed by automated in-line high-performance liquid chromatography-electrospray mass spectrometry (LC-ESMS) analysis and subjected to cell viability screening using a nonradioactive cell proliferation assay. The new prescreening methodology proves to be a powerful tool for establishing structure-activity relationships and for identifying target compounds.

Analysis of two new degradation products of arsenic triglutathione in aqueous solution

Inorganic arsenicals, including arsenite (AsIII) and arsenate (AsV), are well-known human carcinogens. Recently, studies have indicated that arsenic triglutathione (As(GS)3) is unstable in an aqueous solution. The present study was designed to evaluate the degradation mechanism of As(GS)3 in an aqueous solution using high-performance liquid chromatography-electrospray ionisation mass spectrometry (HPLC-ESI-MS). Based on the fragments obtained from MS2 and MS3, we identified two new compounds: one was an isomer of glutathione (GSH), and the other was a product from the cleavage of the glutamyl of oxidised glutathione (GSSG). The isomerization of GSH resulted in the loss of its function such as detoxification of many reactive metabolites. The formation of the two new compounds affected the ratio of GSH/GSSG, and thus may affect the antioxidant and detoxification of GSH/GSSG in mammalian cells.

Enzyme-assisted extraction of antioxidative phenolics from grape (Vitis vinifera L.) residues

Agro-industrial byproducts represent a serious environmental problem and the industries producing these residual materials have incurred expenses for their proper disposal and generally increase the pollution due to the high content of organic substances and might represent legal problems. However, the residues such as grape wastes are potential source of phenolic compounds which are widely known for their high antioxidant activity. Bioprocesses such as enzyme technology represent an alternative for production of those bioactive compounds from agro-industrial byproducts. In this study, different types of commercial enzymes such as Celluclast® 1.5 L, Pectinex® Ultra and Novoferm® were used to release phenolic compounds from grape wastes. The hydrolysates were analyzed in their total phenolic compounds and antioxidant activity with Folin–Ciocaletu test and DPPH· radical-scavenging assay, respectively. A good correlation was obtained between antioxidant activity and phenolics released. The highest antioxidant activities registered were 86.8 ± 0.81, 82.9 ± 0.31 and 90 ± 0.37 % at 12 h for Celluclast® 1.5 L, Pectinex® Ultra and Novoferm®, respectively. Novoferm® had the strongest effect on phenolic release from grape waste, followed by Pectinex® Ultra and Celluclast® 1.5 L. High performance liquid chromatography–electrospray–mass spectrometry clearly revealed that the increment of antioxidant activity is associated with the release of O-coumaric acid.

Transcription factor proteomics: Identification by a novel gel mobility shift-three-dimensional electrophoresis method coupled with southwestern blot and high-performance liquid chromatography–electrospray-mass spectrometry analysis

Transcription factor (TF) purification and identification is an important step in elucidating gene regulatory mechanisms. In this study, we present two new electrophoretic mobility shift assay (EMSA)-based multi-dimensional electrophoresis approaches to isolate and characterize TFs, using detection with either southwestern or western blotting and HPLC–nanoESI-MS/MS analysis for identification. These new techniques involve several major steps. First, EMSA is performed with agents that diminish non-specific DNA-binding and the DNA–protein complex is separated by native PAGE gel. The gel is then electrotransferred to PVDF membrane and visualized by autoradiography. Next, the DNA–protein complex, which has been transferred onto the blot, is extracted using a detergent-containing elution buffer. Following detergent removal, concentrated extract is separated by SDS-PAGE (EMSA-2DE), followed by in-gel trypsin digestion and HPLC–nanoESI-MS/MS analysis, or the concentrated extract is separated by two-dimensional gel electrophoresis (EMSA-3DE), followed by southwestern or western blot analysis to localize DNA binding proteins on blot which are further identified by on-blot trypsin digestion and HPLC–nanoESI-MS/MS analysis. Finally, the identified DNA binding proteins are further validated by EMSA-immunoblotting or EMSA antibody supershift assay. This approach is used to purify and identify GFP-C/EBP fusion protein from bacterial crude extract, as well as purifying AP1 and CEBP DNA binding proteins from a human embryonic kidney cell line (HEK293) nuclear extract. AP1 components, c-Jun, Jun-D, c-Fos, CREB, ATF1 and ATF2 were successfully identified from 1.5 mg of nuclear extract (equivalent to 3 × 10 7 HEK293 cells) with AP1 binding activity of 750 fmol. In conclusion, this new strategy of combining EMSA with additional dimensions of electrophoresis and using southwestern blotting for detection proves to be a valuable approach in the identification of transcriptional complexes by proteomic methods.

Synthesis, Characterization and Optical Properties of a Novel Piperazine Derivative Microcrystal

A novel piperazine derivative (4, 4'-(1, 4-piperazinediyl) bis (4-oxo-2-butenoic acid),PBBA) was successfully synthesized under mild conditions by using piperazine and maleic anhydride as feedstock. Its molecular weight and structure were characterized through High-performance liquid chromatography-electrospray mass spectrometry (HPLC-EMS) and Nuclear magnetic resonance (NMR). Then its especial micrystal was also obtained by recrystallizing through co-precipitation in dimethyl sulfoxide-ethanol system. Scanning electron microscopy (SEM) and optical microscope revealed that the uniform shape microcrystal, which length ranging from 5 μm to 50 μm and width ranging from 2 μm to 15 μm, have unusual optical properties that lights only can be refracted through the middle of the microcrystal, maybe due to its special molecule structure.

A Sensitive HPLC-ESI-MS-MS Method for the Determination of Cotinine in Urine

A simple, sensitive, selective, and reproducible method based on high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry was developed for the determination of nicotine and its major metabolite cotinine in human urine. The internal standard (acetaminophen) was separated from cotinine on a Hypersil Gold C(18) column with retention times of 9.3 and 13.0 min, respectively. The mobile phase consisted of a mixture of 10 mM acetate buffer (pH 5.4) and methanol (45:55, v/v), running through the column at a flow rate of 0.6 mL/min. The chromatographic analysis was operated at 25 degrees C. Sample preparation was prepared by liquid-liquid extraction with a mixture of methyl-t-butyl ether in dichloromethane (1:1, v/v). The precision of the method based on within-day repeatability and reproducibility (day-to-day variation) was below 15% (% coefficient of variation). Good accuracy was observed for both the intra-day or inter-day assays. Limit of quantification was accepted as 0.02 ng using 100 mL samples. The mean recoveries for cotinine and the internal standard were greater than 90%. The method has been applied to the investigation of a 2-h urinary excretion of cotinine in 154 healthy non-smoking Thai volunteers (aged 18-45 years) following the administration of a half-piece (2 mg) of nicotine gum.

Quantitative analysis of the major constituents of St John's wort with HPLC-ESI-MS

A method was developed to profile the major constituents of St John's wort extracts using high-performance liquid chromatography-electrospray mass spectrometry (HPLC-ESI-MS). The objective was to simultaneously separate, identify and quantify hyperforin, hypericin, pseudohypericin, rutin, hyperoside, isoquercetrin, quercitrin and chlorogenic acid using HPLC-MS. Quantification was performed using an external standardisation method with reference standards. The method consisted of two protocols: one for the analysis of flavonoids and glycosides and the other for the analysis of the more lipophilic hypericins and hyperforin. Both protocols used a reverse phase Luna phenyl hexyl column. The separation of the flavonoids and glycosides was achieved within 35 min and that of the hypericins and hyperforin within 9 min. The linear response range in ESI-MS was established for each compound and all had linear regression coefficient values greater than 0.97. Both protocols proved to be very specific for the constituents analysed. MS analysis showed no other signals within the analyte peaks. The method was robust and applicable to alcoholic tinctures, tablet/capsule extracts in various solvents and herb extracts. The method was applied to evaluate the phytopharmaceutical quality of St John's wort preparations available in the UK in order to test the method and investigate if they contain at least the main constituents and at what concentrations.

Rapid and Sensitive High-Performance Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometry for the Quantification of Potassium Dehydroandrographolidi Succinas and Identification of its Degradation Products in Potassium Dehydroandrographolidi Succinas Injection

A high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI- MS/MS) method was established for the quantitative and qualitative analysis of potassium dehydroandrographolidi succinas (DAS-K) and its degradation products, respectively, in DAS-K injection, a widely used drug in China. The analytical HPLC was performed on a Waters RP-C18 column using 5 micromol m(-1) ammonium acetate (pH 3.0, adjusted by formic acid) and methanol as the mobile phase. Detection was performed on a Micromass Quattro micro triple quadrupole mass spectrometer with positive electrospray ionization. Three major degradation products were separated, detected and identified simultaneously by HPLC-ESI-MS/MS results. An HPLC method for the assay of DAS-K in its injection was then established. The multiple reaction monitoring function (m/z 550.14-->m/z 297.04 for DAS-K) enabled the quantification of DAS-K down to 0.2 ng m(-1). The calibration graph was linear (r better than 0.999, n = 7) in the concentration range 2.0-100 ng mL(-1). The values for the intra- and inter-day relative standard deviation (RSD) were less than 1.7% and 2.5% for DAS-K. The average recovery rate was over 98%, with an RSD less than 3.0%. This is the first report on the degradation products in DAS-K injection.

Simultaneous Analysis of Selected Typical Antibiotics in Manure by Microwave-Assisted Extraction and LC–MS n

Analysis of antibiotics in the environment has become an urgent issue. A novel method entailing microwave-assisted extraction (MAE) and high-performance liquid chromatography–electrospray mass spectrometry (LC–MSn) has been developed for determination of selected typical antibiotics, including quinolones, sulfonamides, and tetracyclines, in manure. Compared with ultrasonic extraction, MAE significantly increased recovery of fluoroquinolones (63–106%), sulfonamides (64–133%), and tetracyclines (64–109%) from manure. Acetonitrile acidified with formic acid buffer solution (pH 4.0) was used for extraction of swine manure whereas chicken manure was extracted with 0.1 M EDTA–McIlvaine buffer solution. Limits of quantification (LOQ) for all compounds were in the range 5.12–168.4 μg kg−1 dry matter, which were satisfactory for analysis of all samples. The suitability of the method was assessed by analysis of manure from six different sites.

Effect of bifendate on the pharmacokinetics of talinolol in healthy subjects

To evaluate the effect of bifendate on the pharmacokinetics of talinolol, 16 unrelated male healthy subjects were selected for this study. After repeated oral administration of placebo or bifendate (three times daily for 14 days), the plasma concentration of talinolol was measured by high-performance liquid chromatography-electrospray mass spectrometry. This study was carried out in a randomized, single-blinded, placebo-controlled, crossover manner.After the treatment of bifendate, the area under the curve (AUC0–∞) was decreased significantly by 11.2% (90% confidence interval (CI), 7.3–12.4%; p = 0.001), and the Cmax value of talinolol was decreased by approximately 9.7% (90% CI, 5.5–11.4%; p = 0.001). The oral clearance of talinolol was increased significantly by 13.1% (90% CI, 8.0–14.4%; p = 0.001).The results suggest that the treatment of bifendate can decrease the plasma concentration of talinolol in healthy subjects.

Determination of Naloxone and Nornaloxone (Noroxymorphone) by High-Performance Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry

A highly sensitive method was developed to measure naloxone and its metabolite nornaloxone in human plasma, urine, and human liver microsomes (HLM). Naltrexone-d(3) and oxymorphone-d(3) were used as respective internal standards. Solid-phase extraction, using mixed mode extraction columns and 0.1 M phosphate buffer (pH 5.9), was combined with high-performance liquid chromatography interfaced by electrospray ionization to tandem mass spectrometry. The calibration range in plasma was 0.025 to 2 ng/mL for naloxone and 0.5 to 20 ng/mL for nornaloxone. It was 10 to 2000 ng/mL in urine and 0.5 to 20 ng/mL in HLM for both. Enzymatic hydrolysis of urine was optimized for 4 h at 40 degrees C. Intra- and interrun accuracy was within 15% of target; precision within 13.4% for all matrices. The mean recoveries were 69.2% for naloxone and 32.0% for nornaloxone. Analytes were stable in plasma and urine for up to 24 h at room temperature and in plasma after three freeze-thaw cycles. In human subjects receiving 16 mg buprenorphine and 4 mg naloxone, naloxone was detected for up to 2 h in all three subjects and up to 4 h in one subject. Mean AUC(0-24) was 0.303 +/- 0.145 ng/mL.h; mean C(max) was 0.139 +/- 0.062 ng/mL; and T(max) was 0.5 h. In 24-h urine samples, about 55% of the daily dose was excreted in either conjugated or unconjugated forms of naloxone and nornaloxone in urine. When cDNA-expressed P450s were incubated with 20 ng of naloxone, nornaloxone formation was detected for P450s 2C18, 2C19, and 3A4. Naloxone utilization exceeded nornaloxone formation for 2C19 and 3A4, indicating they may produce products other than nornaloxone. These results demonstrate a new method suitable for both in vivo and in vitro metabolism and pharmacokinetic studies of naloxone.