Propofol Glucuronide Research Articles

Rapid Evaluation Method for Propofol Abusing with Hair by Using Centrifugal Filter and Liquid Chromatography-tandem Mass Spectrometry

Background: Since propofol is rapidly metabolized and excreted from the body, it is not easy to quantify its intake in blood or urine samples over the time. In this case, the hair sample would be more advantageous to estimate during the abuse period. However, the presence of protein and lipid in the hair sample could interfere with extraction and be problematic during mass spectrometric analysis. Objective: The aim of this study is to develop a simple and less time-consuming method for the extraction of propofol glucuronide by removing hair interferences with the centrifugal filter. Methods: Hair samples were washed and dissolved with sodium hydroxide solution. This dissolved hair solution was applied to the centrifugal filter and centrifuged. The filtrate was extracted with ethyl acetate and evaporated to dryness. The residue was reconstituted with methanol and analyzed by liquid chromatography coupled with tandem mass spectrometry. This developed analytical method was validated by testing linearity, selectivity, accuracy, precision, recovery, matrix effect and stability of propofol glucuronide. Results: The validation results showed good linearity over the concentration range of 0.5~500 pg/mg, with a correlation coefficient of 0.9991. The LOD and LLOQ were 0.2 and 0.5 pg/mg, respectively. The intra-and inter-day precision and accuracy were acceptable within 14.5% for precision and 10.1% for accuracy. Similarly, the developed method revealed high sample recovery (>88%), low hair matrix effect (<10%) and highly-efficient extraction procedure. Conclusion: This well-validated procedure was successfully applied to determine propofol glucuronide in a rat hair sample and can be applicable, with high potential, in the field of forensic toxicology especially with increasing abuse and accidental overdose of propofol.

Development of a LC–MS/MS method for determination of propofol-glucuronide in hair and preliminary study on relationships between dose and hair concentration

Propofol abuse has been reported worldwide, suggesting the need to establish analytical methods for human biological samples to investigate the abuse of propofol. This study aimed to investigate the relationship between dose and hair concentration using a simple and rapid analytical method developed and validated in this study. In the sample preparation, hair samples were washed with distilled water and methanol and extracted in methanol during 16h at room temperature. After centrifugation and evaporation, the residue was reconstituted and filtered through a 0.22μm membrane filter before LC–MS/MS analysis. The precursor-to-product ion transitions were 353 → 175, 113 for propofol glucuronide and m/z 370 → 175, 113 for internal standard(propofol glucuronide-d17). The calibration curves were satisfactory (R2=0.9997) and the limits of detection and quantification were 2 and 5pg/mg, respectively. In addition, this study collected the history of propofol use from subjects using a questionnaire and analyzed subjects' hair samples using a validated analytical method. As a result, the concentrations of propofol glucuronide ranged from 7 to 122pg/mg (mean : 51pg/mg). There were cases of positive relationships, but generally there was no correlation between dose and hair concentration.

Propofol and propofol glucuronide concentrations in hair following medical propofol administration and in forensic death cases

Propofol is the most preferred drug for general anesthesia as well as for analgosedation. However, the rate of abuse cases has increased in the past decade. Hair analysis is considered as the method of choice to determine chronic drug use, and propofol and propofol glucuronide have already been used to confirm previous propofol administration. However, given its frequent medical use, it is important that nonmedical propofol abuse can be distinguished from medical propofol application. Nineteen hair samples collected from living subjects who received different doses of propofol in the setting of medical treatment and 31 hair samples from forensic death cases with indications of previous propofol administration were examined using our previously described method enabling the simultaneous extraction of propofol and propofol glucuronide from hair followed by validated liquid chromatography–tandem mass spectrometry analyses. Recent propofol administration was verified for eight of 19 living cases and 29 of 31 deceased cases. Of the living cases, propofol glucuronide could be detected in all eight cases, whereas propofol could only be detected in three of these cases. Propofol glucuronide could be detected more frequently and in higher concentrations than propofol following medical propofol administration and observed concentrations varied more widely. Although further research is still required to clarify the mechanisms involved in propofol incorporation into hair and to establish reliable cutoff concentrations for the differentiation of medical from nonmedical propofol use, it seems likely that relatively high concentrations of propofol found across multiple hair segments strongly suggest a nonmedical propofol abuse.

Simultaneous extraction of propofol and propofol glucuronide from hair followed by validated LC–MS/MS analyses

Besides its clinical application, the anaesthetic agent propofol is being increasingly misused, mostly by healthcare professionals, and its abuse potential gained worldwide attention after the tragic death of Michael Jackson in 2009. Due to the short duration of its narcotic effects, propofol abuse is especially easy to hide compared with the use of other recreational drugs. However, propofol possesses a very narrow therapeutic window between the desired effect and potentially fatal toxicity, making abuse of the drug extremely dangerous even in experienced physicians. Consequently, it is important that forensic laboratories possess a sensitive and specific method for the detection of chronic propofol abuse. We present a simple, fast and reliable method to simultaneously extract propofol and its main metabolite propofol glucuronide from hair, followed by sensitive LC–MS/MS analyses, allowing to determine a chronic propofol abuse. Difficulties regarding the detection of propofol using LC–MS/MS were solved by using a derivatization reaction with 2-fluoro-1-methylpyridinium-p-toluene-sulfonate and triethylamine. Reliability of extraction method and subsequent LC–MS/MS analyses was confirmed under consideration of the validation parameters selectivity, linearity, accuracy and precision, analytical limits, processed sample stability, matrix effects and recovery. Appropriate quantification (LLOQ=10pg/mg hair) and detection limits (3.6pg/mg hair for propofol and 7.8 pg/mg hair for propofol glucuronide) could be achieved, enabling to detect even small amounts of both analytes. Applicability of the method was confirmed by analysis of three human hair samples from deceased with suspicion of chronic propofol abuse.

Verification of propofol sulfate as a further human propofol metabolite using LC-ESI-QQQ-MS and LC-ESI-QTOF-MS analysis.

Propofol (2,6-diisopropylphenol) is a water-insoluble, intravenous anesthetic that is widely used for the induction and maintenance of anesthesia as well as for endoscopic and pediatric sedation. After admission, propofol undergoes extensive hepatic and extrahepatic metabolism, including direct conjugation to propofol glucuronide and hydroxylation to 2,6-diisopropyl-1,4-quinol. The latter substance subsequently undergoes phase II metabolism, resulting in the formation of further metabolites (1quinolglucuronide, 4quinolglucuronide and 4quinol-sulfate). Further minor phase I propofol metabolites (2-(ω-propanol)-6-isopropylphenol and 2-(ω-propanol)-6-isopropyl-1,4-quinol)) are also described. Due to its chemical structure with the phenolic hydroxyl group, propofol is also an appropriate substrate for sulfation by sulfotransferases. The existence of propofol sulfate was investigated by liquid chromatography electrospray ionization triple quadrupole mass spectrometry (LCESIQQQ-MS) and liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry (LCESI-QTOF-MS). A propofol sulfate reference standard was used for identification and method development, yielding a precursor at m/z 257 (deprotonated propofol sulfate) and product ions at m/z 177 (deprotonated propofol) and m/z 80 ([SO3]-). Propofol sulfate - a further phase II metabolite of propofol - was verified in urine samples by LC-ESI-QQQ-MS and LC-ESI-QTOF-MS. Analyses of urine samples from five volunteers collected before and after propofol-induced sedation verified the presence of propofol sulfate in urine following propofol administration, whereas ascertained concentrations of this metabolite were significantly lower compared with detected propofol glucuronide concentrations. The existence of propofol sulfate as a further phase II propofol metabolite in humans could be verified by two different detection techniques (LCESIQQQ-MS and LC-ESI-QTOFMS) on the basis of a propofol sulfate reference standard. Evaluation of the quantitative analyses of propofol sulfate imply that propofol sulfate represents a minor metabolite of propofol and is only slightly involved in human propofol clearance.

Determination of propofol glucuronide from hair sample by using mixed mode anion exchange cartridge and liquid chromatography tandem mass spectrometry.

The main objective of this study was to develop and validate a simpler and less time consuming analytical method for determination of propofol glucuronide from hair sample, by using mixed mode anion exchange cartridge and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The study uses propofol glucuronide, a major metabolite of propofol, as a marker for propofol abuse. The hair sample was digested in sodium hydroxide solution and loaded in mixed-mode anion cartridge for solid phase extraction. Water and ethyl acetate were used as washing solvents to remove interfering substances from the hair sample. Consequently, 2% formic acid in ethyl acetate was employed to elute propofol glucuronide from the sorbent of mixed-mode anion cartridge, and analyzed by LC-MS/MS. The method validation parameters such as selectivity, specificity, LOD, LLOQ, accuracy, precision, recovery, and matrix effect were also tested. The linearity of calibration curves showed good correlation, with correlation coefficient 0.998. The LOD and LLOQ of the propofol glucuronide were 0.2 pg/mg and 0.5 pg/mg, respectively. The intra and inter-day precision and accuracy were acceptable within 15%. The mean values of recovery and matrix effect were in the range of 91.7-98.7% and 87.5-90.3%, respectively, signifying that the sample preparation, washing and extraction procedure were efficient, and there was low significant hair matrix effect for the extraction of propofol glucuronide from hair sample on the mixed mode anion cartridge. To evaluate the suitability of method, the hair of propofol administered rat was successfully analyzed with this method.

P2 The detection of propofol glucuronide in human hair using LC-MS/MS: Case study and survey of high-risk health professionals

Background Propofol (2,6-diisopropylphenol) is a commonly used agent for general anesthesia because of its short duration, quick recovery time, and minimal side effects. Although not a controlled substance in the United States, propofol abuse and misuse by medical professionals has increased 5-fold between 1997 and 2011. Propofol detection in biological fluids has a very short detection window and the presence of propofol in hair does not distinguish between ingestion and environmental exposure. A metabolite, propofol glucuronide, is formed in the liver and may be detected in the hair. The purpose of this study is to validate a method for the detection of propofol glucuronide in hair and apply this method to a case study and a survey of high-risk health professionals. Methods A method for the detection of propofol glucuronide was developed and validated according commonly recognized guidelines. Briefly, hair specimens were powdered and extracted into water while incubating in a sonicating water batch without heat over night. Extracts were subjected to solid phase extraction using quaternary amine with chloride counter ion extraction cartridges. The eluates were evaporated under nitrogen at 40 °C, reconstituted in 100 μL of DI water. Extracts were separated using Synergy Polar RP column (50mm x 2mm x 2.5 μm particle size) on an Agilent 1200 HPLC system. The compounds were detected on an AB Sciex 5500 tandem mass spectrometer (LC-MS/MS) following the transitions of 353,1 m/z to 176,7 m/z , 112,6 m/z , 84,7 m/z for propofol glucuronide and 370,1 m/z to 193,7 m/z and 369,7 m/z for the internal standard, propofol glucuronide- d 17 . A hair specimen that originated from a 50 year old woman that was anesthetized for 15 minutes was analyzed. Additionally, a survey of 300 de-identified hair specimen remnants that originated from health professionals undergoing treatment for substance abuse. Results The empirically determined LOD and LOQ for this assay were 4 pg/mg and 8 pg/mg, respectively. The intra- and inter-assay bias and imprecision studies were all acceptable ( Conclusion This method demonstrated sufficient precision, accuracy and robustness with an analytical measurement range between 8 pg/mg and 100 pg/mg. While being sensitive enough to identify two individuals in a survey of high-risk substance abuse treatment participants, the method was not able to identify propofol metabolite in the hair of a woman receiving a routine quantity of propofol administered during a short surgical procedure. This method provides substance abuse treatment professionals with another objective tool to identify propofol abusers.

Rapid and sensitive determination of propofol glucuronide in hair by liquid chromatography and tandem mass spectrometry

A fast, sensitive and selective liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the detection and quantitation of propofol glucuronide in human hair has been developed and validated. Propofol glucuronide was extracted from 10mg of hair using a simple methanol extraction method, with recovery greater than 91% at 3 quality control samples (15, 100, 4000pg/mg). A reversed phase column (C8) was used to analyze and the mobile phase was composed of ammonium formate and acetonitrile gradient at a flow rate of 0.2mL/min. The lower limit of quantitation (LLOQ) was 5pg/mg and the assay was linear to 5000pg/mg. The intra- and inter-day precision (% CV, coefficient of variation) ranged from 1.26 to 4.50% while the accuracy (% RE, relative error) were -4.24 to 4.4%. The matrix effects were monitored at 3 different concentrations and the %CV of the results for these concentrations was less than 10.6%. Propofol glucuronide was stable during processing and analysis in human hair. The procedure was validated and applied to the analysis of hair samples in human subjects previously administered in propofol.

Sex Difference in Formation of Propofol Metabolites: A Replication Study

Women recover faster from propofol anaesthesia and have been described to have a higher incidence of awareness during surgery, compared to men - an effect that may be inherent in sex differences in propofol metabolism. In an observational study, 98 ASA I-II patients treated with continuous propofol infusion were recruited. The associations between sex and CYP2B6 and UGT1A9 polymorphisms with dose- and weight-adjusted area under the total plasma level time curves (AUC) for propofol, and its metabolites propofol glucuronide (PG), 4-hydroxypropofol (OHP) and hydroxyl glucuronide metabolites 4-hydroxypropofol-1-O-β-D-glucuronide (Q1G) and 4-hydroxypropofol-4-O-β-D-glucuronide (Q4G), were analysed. Significantly higher AUC of PG (1.3 times, p=0.03), Q1G (2.9 times, p<0.001), Q4G (2.4 times, p<0.01) and OHP (4.6 times, p=0.01) were found in women (n=53) than in men (n=45) after intravenous infusion of propofol using target-controlled infusion system. There was, however, no significant impact of gene polymorphisms on propofol biotransformation. The results, which are supported by a previous pilot study using a propofol bolus dose, suggest that, compared to men, more rapid propofol metabolism may occur in women - a factor that may contribute to the mentioned differences in the efficacy of propofol anaesthesia between male and female patients.

Long-Term Detection of Propofol Glucuronide in Urine Following Anesthetic Induction and Maintenance with Propofol

Propofol is the most commonly used compound for the intravenous induction and maintenance of anesthesia. Propofol addiction and abuse have become causes for concern in the healthcare community, especially among anesthesia and surgical professionals. The US Drug Enforcement Administration does not list propofol on any Schedules and most hospitals do not have inventory controls in place to prevent its misuse. Propofol is detectable in blood plasma as the parent compound for as much as 15 hours post-anesthesia. The metabolite propofol glucuronide (PPFG) has been detected in blood and urine as far out as 60 hours. Here we report the long-term renal excretion of PPFG in specimens from A) four participants following a 14-day course of orally ingested propofol dosing, and B) a female patient following anesthetic induction and 15 minutes’ maintenance with propofol. Urinary PPFG was measurable well above limits of quantitation up to 6 days following oral ingestion and 28 days post-anesthesia. We also present a third set of data evaluating the likelihood of passive exposure to aerosolized propofol in the surgical environment by analyzing the levels of urinary PPFG of healthcare workers following operating room work shifts. The results presented here demonstrate that quantitation of PPFG in urinary samples is an efficient method of long-term screening for propofol misuse and abuse.

Urinary metabolites after intravenous propofol bolus in neonates

In neonates, propofol mainly undergoes hydroxylation to quinol metabolites with only limited glucuronidation. The aim of this study is to search for covariates of neonatal propofol biotransformation based on 24h urine collections. In neonates receiving an intravenous propofol bolus for short procedural sedation, urine was collected during 24h. Urinary propofol metabolites [propofol glucuronide (PG), 1- and 4-quinol glucuronide (QG)] were determined using high-performance liquid chromatography after a dual-step solid phase extraction combined with ultraviolet and fluorescence detection. Propofol metabolites, their contribution to total metabolite elimination and propofol glucuronide/quinol glucuronide (PG/QG) ratio were determined. The impact of continuous [postmenstrual age (PMA), postnatal age (PNA), body weight, propofol dose, creatinaemia] and dichotomous variables [PNA≤7days (yes/no), PNA≥10days (yes/no), hyperbilirubinaemia (yes/no), cardiopathy (yes/no)] on PG/QG ratio and on patients with low (≤10%) vs. high (>10%) urinary PG recovery were examined. Thirty-two neonates were included. Median total propofol metabolite recovery was 40.95 (2.01-129.81)% with PG/QG ratio 0.44 (0.01-5.93). PNA (dichotomous 7days as well as 10days) was a significant covariate of PG/QG ratio. Late PNA more frequently resulted in high urinary PG fraction. Significance was more pronounced with PNA 10days as cut-off point for early neonatal life compared to 7days. Age 10days is pivotal in early life propofol metabolism. This confirms earlier documented propofol clearance studies. This is the first report of the modified quantification assay used to determine urinary propofol metabolites in neonates.

Comparison of GC/MS and LC/MS methods for the analysis of propofol and its metabolites in urine

Gas chromatography–mass spectrometry (GC/MS) and liquid chromatography–mass spectrometry (LC/MS) were compared for their capacity to metabolite identification, sensitivity, and speed of analysis for propofol and its metabolites in urine samples. Acidic hydrolysis, liquid–liquid extraction (LLE), and trimethylsilyl (TMS) derivatization procedures were applied for GC/MS analysis. The LC/MS analysis used a simple sample pretreatment based on centrifugation and dilution. Propofol and four metabolites were successfully analyzed by GC/MS following TMS derivatization. One compound, di-isopropanolphenol was tentatively characterized as a new metabolite observed for the first time in human urine. The TMS derivatization greatly improved the chromatographic properties and detection sensitivity, especially for hydroxylated metabolites. The lower limits of quantitation (LLOQ) of propofol were about 325 and 0.51ng/mL for the GC/MS scan mode and selected ion monitoring (SIM) mode, respectively. In addition, five conjugated propofol metabolites were successfully analyzed by LC–MS/MS in negative ion mode. The detection sensitivity for these conjugated metabolites could be greatly enhanced by the addition of triethylamine to the mobile phase without any loss of LC resolution capacity. The LLOQs of propofol-glucuronide (PG) were about 1.17 and 2.01ng/mL for the LC–MS-selected ion monitoring (SIM) and multiple reaction monitoring (MRM) mode, respectively. Both GC/MS and LC/MS methods sensitively detected nine metabolites of propofol and could be used to provide complementary data for the reasonable propofol metabolism study. Urinary excretion profiles for propofol and its metabolites following administration to human were suggested based on the total ion chromatograms obtained by GC/MS and LC/MS methods, respectively.

Influence of sex on propofol metabolism, a pilot study: implications for propofol anesthesia

The basis of high intersubject variability of propofol metabolism is unclear. Therefore, we examined the influence of genetic polymorphisms of the key metabolizing enzymes cytochrome P450 2B6 (CYP2B6) and uridine diphosphate (UDP)-glucuronosyltransferase 1A9 (UGT1A9), age, and sex on propofol biotransformation in vitro and in vivo. Plasma concentrations of propofol, 4-hydroxypropofol, and their glucuronides were measured over 20 min in 105 patients after a single intravenous bolus of propofol. Propofol 4-hydroxylation activity, genotypes, and content of CYP2B6 protein in 68 human livers were determined. The common single nucleotide polymorphisms (SNPs) for the CYP2B6 and UGT1A9 genes were analyzed by polymerase chain reaction (PCR). Plasma levels of propofol metabolites showed high interindividual variability (range of coefficient of variation 89-128%). This was supported by in vitro data showing similar variability of propofol 4-hydroxylation in liver microsomes and 1.9-fold higher CYP2B6 protein content in the livers from women. No significant relationships were revealed between the SNPs studied and propofol metabolism. However, patients' sex had a pronounced effect on propofol metabolism. Thus, women had higher amounts of propofol glucuronide (1.25-fold; p = 0.03), 4-hydroxypropofol-1-glucuronide (2.1-fold; p = 0.0009), and 4-hydroxypropofol-4-glucuronide (1.7-fold; p = 0.02) as shown by the weight-corrected area under the time-plasma concentration curve of metabolites. Additionally, the sexual dimorphism in 4-hydroxypropofol glucuronidation was prominent in the 35- to 64-year-old subgroup. No significant effects of CYP2B6 and UGT1A9 SNPs or age on propofol metabolism were revealed in this pilot study, but there was a pronounced effect of sex, a finding that indicates an important factor for the previously described sex difference in systemic clearance of propofol seen.

Interspecific Difference Assay of UDP-glucuronosyltransferase 1A9 Activities in Liver Microsomes by Ultra-performance Liquid Chromatography-tandem Mass Spectrometry

Aim To establish a UPLC-ESI-MS method for the assay of UDP-glucuronosyltransferases(UGT) 1A9 activities in human and animal liver microsomes, to investigate the interspecific differences of UGT1A9. Methods The probe substrate of propofol ( m/ z 177.3), II phase metabolite of propofol glucuronide (PG) ( m/ z 377.3, [M + Na] +), and internal standard (IS) of 3-acetamidophenol ( m/ z 152.2, [M + H] +) were quantified by selective ion monitor (SIM), using electrospray ionization-MS (ESI-MS), acquity UPLC BEH C 18 column (50 mm × 2.1 mm, 1.7 μm) and a mixture of acetonitrile(B)-0.5% acetic acid(A) as flow phase in gradient elution at flow rate of 0.3 mL·min −1. Result The linearity was within 0.2 to 12.8 μg·mL −1 for PG. The intra- and inter-day precisions were less than 3% and the accuracies ranged from −3.46% to 3.03%. The recoveries of PG from human liver microsomes (HLMs) were 96.5%-98.6%. The method was successfully used to determine the kinetics of propofol glucuronidation in human and different animals liver microsomes. The results indicated that the kinetics of propofol O-glucuronidation in liver micro-somes from human, mouse, rabbit and sheep were fitted to the substrate inhibition equation, whereas the kinetics in rat liver micro-somes was fitted to the two-enzyme Michaelis–Menten equation. The intrinsic clearance ( CL int ) of propofol glucuronidation in different liver microsomes was in the order of mice > rats ( CL int1 + CL int2 )>sheep>rabbits>human. Conclusion The method is rapid, sensitive and selective for assay of UGT1A9 activities in liver microsomes. The results indicated that the kinetics of propofol O-glucuronidation in rat liver microsomes is different from that in HLMs, and the CL int value for propofol O-glucuronidation in liver microsomes from mice, sheep, and rabbit are remarkably higher than that in HLMs. In animal experiments for drug development, these interspecific differences should be taken into consideration.

Urinary propofol metabolites in early life after single intravenous bolus

BackgroundPropofol clearance is lower in neonates than in adults and displays extensive interindividual variability, in part explained by postmenstrual age (PMA) and postnatal age (PNA). Since propofol is almost exclusively cleared metabolically, urinary propofol metabolites were determined in early life and compared with similar observations reported in adults. MethodsTwenty-four hours urine collections were sampled after a single i.v. bolus of propofol (3 mg kg−1) in neonates undergoing procedural sedation. Clinical characteristics (PMA, PNA, weight, and cardiopathy) were recorded. Urine metabolites [propofol glucuronide (PG), 1- and 4-quinol glucuronide (QG)] were quantified using high-pressure liquid chromatography. Urine recovery (% administered dose) and the contribution of PG and QG to urinary elimination were calculated. Data were reported by median and range, analysed by Mann–Whitney U or Spearman's rank. ResultsEleven neonates (median PNA 11 days, PMA 38 weeks) were included. Median propofol metabolite recovery was 64% (range 34–98%). PG contributed 34% (range 8–67%) and QG 65% (range 33–92%). There was no significant correlation between either PMA, PNA, or cardiopathy and propofol metabolites. Compared with adults, the contribution of PG (34% vs 77%) was lower and the contribution of QG (65% vs 22%) was higher in neonates. ConclusionsPropofol metabolism in neonates differs from adults, reflecting the age-dependent limited glucuronidation capacity. Hydroxylation to quinol metabolites already contributes to propofol metabolism. These differences likely explain the PMA- and PNA-dependent reduced propofol clearance in neonates.

Quantitative measurement of propofol and in main glucuroconjugate metabolites in human plasma using solid phase extraction–liquid chromatography–tandem mass spectrometry

A high-performance liquid chromatographic method coupled with tandem mass spectrometry detection has been developed for the determination of propofol and its main glucuroconjugate metabolites (propofol–glucuronide (PG), 1-quinol-glucuronide (1-QG) and 4-quinol-glucuronide (4-QG) in human plasma. All compounds were extracted with a single solid phase extraction procedure using Max Oasis © cartridges. Propofol and thymol (internal standard) were analyzed using a C8 reversed-phase column with a mobile phase consisting of methanol–water (75:25, v/v) containing 0.025% NH 4OH. Chromatography of glucuroconjugate metabolites and phenyl-β- d-glucuronide (internal standard) was performed using a hydrophilic interaction liquid chromatography (HILIC) and a mixture of acetonitrile/water/ammonium acetate buffer (100 mM, pH 5, 87/1/12, v/v/v). Both chromatographic separations were achieved in isocratic mode allowing a rapid analysis without re-equilibration of the phase. The method is specific and sensitive with a range of 10–1500 ng mL −1 for propofol and 1-QG, 20–3000 ng mL −1 for PG and 25–3750 ng mL −1 for 4-QG. The regression curves were linear for all compounds. The method is accurate and precise with intra-assay and inter-assay precision <8% and bias ≤6% for all compounds. This assay has allowed the successful measurement of propofol and its main glucuroconjugate metabolites in human plasma from 24 patients undergoing anaesthesia for elective partial hepatectomy surgery.

Relative antioxidant capacities of propofol and its main metabolites.

The antioxidant activity of propofol, a widely used anesthetic, has previously been demonstrated, but no study has focused on propofol metabolites although propofol undergoes extensive metabolism. In the present study, the antioxidant properties of propofol and its metabolites were studied by measuring malondialdehyde (MDA) produced from lipid peroxidation by microsomes triggered with several free radical generating systems. True MDA determination was performed using a specific high performance liquid chromatography technique. Gas chromatography-isotope ratio mass spectrometry methodology was also used to assess the antioxidant action in a homogeneous aqueous environment. Propofol, 2,6-di-isopropyl-1,4-quinol (1,4-quinol) metabolite and 3,5-di- tert-butyl-4-hydroxytoluene markedly inhibit lipid peroxidation at concentrations lower than 5 microM. The binding of the glucuroconjugated moiety to either one of two hydroxyl groups of 1,4-quinol lowers the radical scavenging activity. Propofol glucuronide did not exert any radical scavenging activity except when peroxidation was induced with tert-butylhydroperoxide. Our data demonstrate that propofol and its metabolites inhibit lipid peroxidation at concentrations similar to those measured in human plasma during anesthesia. Their antioxidant efficiency is influenced by several factors, including the type of radical initiator involved and the site of radical production.

Propofol metabolites in man following propofol induction and maintenance

The pharmacokinetics of propofol in man is characterized by a rapid metabolic clearance linked to glucuronidation of the parent drug to form the propofol-glucuronide (PG) and sulfo- and glucuro-conjugation of hydroxylated metabolite via cytochrome P450 to produce three other conjugates. The purpose of this study was to assess the urine metabolite profile of propofol following i.v. propofol anaesthesia in a Caucasian population. The extent of phase I and phase II metabolism of propofol was studied in 18 female and 17 male patients after an anaesthesia induced and maintained for at least 4 h with propofol. The infusion rates (mg kg(-1) h(-1)) of propofol were (mean (SD)) 4.1 (1.0) and 4.5 (1.3) for males and females, respectively. Urine was collected from each patient for the periods 0-4, 4-8, 8-12, and 12-24 h after the start of propofol administration. In a preliminary study, the three main glucuro-conjugated metabolites were isolated from urine and characterized by magnetic resonance spectroscopy. The quantification of these metabolites for the different collection periods was then performed by a HPLC-UV assay. Total recovery of propofol in the metabolites studied amounts to 38%, of which 62% was via the PG metabolite and 38% via cytochrome P-450. This percentage is significantly higher than that previously reported from patients after a bolus dose of propofol. Extreme values for PG (0-24 h period) were included from 73 to 49%. There was no significant difference between female and male patients in the metabolite ratio. We conclude that the extent of hydroxylation in propofol metabolism was higher than in previous findings after administration of anaesthetic doses of propofol. Moreover, the ratio between hydroxylation and glucuronidation of propofol is subject to an inter-patient variability but this does not correlate with the dose of propofol. However, the variation of the metabolite profile observed in the present report does not seem to indicate an extended role of metabolism in pharmacokinetic variability.

Biosynthesis of drug glucuronides for use as authentic standards

Introduction: Glucuronidation by the uridine diphosphate glucuronosyltransferases (UGTs) plays a pivotal role in the clearance mechanism of both xenobiotics and endobiotics. The detection of glucuronides at low micromolar concentrations is required to accurately model in vitro enzyme kinetics and in vivo pharmacokinetics. However, relatively few glucuronides are currently available as standards for developing liquid chromatography and mass spectroscopy (LC/MS) bioanalytical methods. Methods: The glucuronidation capacity of hepatic microsomes prepared from rat (RLM), dog (DLM), monkey (MLM), and human (HLM) was examined for five xenobiotic substrates. In each case, glucuronide standards were produced using the enzyme source most efficient for the production of that specific glucuronide. Results: Dog hepatic microsomes were used to produce glucuronides for anthraflavic acid (yield: 14 mg), buprenorphine (yield: 14 mg), and octylgallate (total yield: 13 mg), whereas propofol glucuronide (yield: 20 mg), and ethinylestradiol glucuronide (yield: 8 mg) were prepared using HLM. All glucuronides were characterized by LC/MS/MS and nuclear magnetic resonance (NMR) spectroscopy. Discussion: The multimilligram quantities of glucuronide standards produced by this method have many applications throughout drug discovery and toxicology. In addition to allowing the quantification of glucuronide formation from in vitro and in vivo studies, the authentic standards produced could also be used to assess potential pharmacological or toxicological effects of metabolites.

Direct high-performance liquid chromatography determination of propofol and its metabolite quinol with their glucuronide conjugates and preliminary pharmacokinetics in plasma and urine of man

Propofol (P) is metabolized in humans by oxidation to 1,4-di-isopropylquinol (Q). P and Q are in turn conjugated with glucuronic acid to the respective glucuronides, propofol glucuronide (Pgluc), quinol-1-glucuronide (Q1G) and quinol-4-glucuronide (Q4G). Propofol and quinol with their glucuronide conjugates can be measured directly by gradient high-performance liquid chromatographic analysis without enzymic hydrolysis. The glucuronide conjugates were isolated by preparative HPLC from human urine samples. The glucuronides of P and Q were present in plasma and urine, P and Q were present in plasma, but not in urine. Quinol in plasma was present in the oxidised form, the quinone. Calibration curves of the respective glucuronides were constructed by enzymic deconjugation of isolated samples containing different concentrations of the glucuronides. The limit of quantitation of P and quinone in plasma are respectively 0.119 and 0.138 μg/ml. The limit of quantitation of the glucuronides in plasma are respectively: Pgluc 0.370 μg/ml, Q1G 1.02 μg/ml and Q4G 0.278 μg/ml. The corresponding values in urine are: Pgluc 0.264 μg/ml, Q1G 0.731 μg/ml and Q4G 0.199 μg/ml. A pharmacokinetic profile of P with its metabolites is shown, and some preliminary pharmacokinetic parameters of P and Q glucuronides are given.