Prediction and Validation of Phase II Glucuronide Conjugates in Urine Using Combined Non-Targeted and Targeted LC–HRMS/MS Workflows and Their Validation for over 200 Drugs

An integrated strategy for in vivo metabolite profiling using high-resolution mass spectrometry based data processing techniques

Platinum-based cytostatic drugs are one of the most widely used cancer treatments. They are excreted via the urinary tract and can reach the environment through wastewater, posing a risk to human health due to their side effects. Four identification and quantification techniques, including liquid chromatography (LC) separation coupled to (i) a diode array ultraviolet (UV(DAD)) (ii), mass spectrometer in single ion monitoring mode (LC-MS) and (iii) multiple reaction monitoring mode (LC-MS/MS) and (iv) derivatization with diethyldithiocarbamate prior to LC-MS/MS analysis, have been optimized and compared for the multiresidue determination of main platinized cytostatic drugs (cisplatin, carboplatin, and oxaliplatin) in urine samples. Parameters that affect the efficiency of the chromatographic separation and analytical determination of different methods (column, mobile phase, wavelength, precursor ions, fragmentor, and product ions) were optimized. Analytical features, such as matrix effect, sensitivity, precision, selectivity, and linearity, were calculated. In terms of selectivity, the derivatization technique was discarded since it was only applicable to the platinated sum. A high dilution of the sample with LC-UV(DAD) was needed to reduce the matrix effect. Overall, the LC-MS/MS method presented the best analytical features (% RSD ≤ 12.8%, R2 ≥ 0.991, or method-detection limits between 0.01-1 µg mL-1). The selected method was applied to the quantification of platinized cytostatic drugs in hospital urine samples from oncologic patients.

Rapid and sensitive determination of coumarin and 7-hydroxycoumarin and its glucuronide conjugate in urine and plasma by high-performance liquid chromatography

PurposeToxicological analyses of biological samples play important roles in forensic and clinical investigations. Ingested drugs are excreted in urine as conjugates with endogenous substances such as glucuronic acid; hydrolyzing these conjugates improves the determination of target drugs by liquid chromatography–tandem mass spectrometry (LC–MS/MS). In this study, we sought to improve the enzymatic hydrolysis of glucuronide conjugates of five psychoactive drugs (11-nor-9-carboxy-Δ9-tetrahydrocannabinol, oxazepam, lorazepam, temazepam, and amitriptyline).MethodsThe efficiency of enzymatic hydrolysis of glucuronide conjugates in urine was optimized by varying temperature, enzyme volume, and reaction time. The hydrolysis was performed directly on extraction columns. This analysis method using LC–MS/MS was applied to forensic autopsy samples after thorough validation.ResultsWe found that the recombinant β-glucuronidase B-One® quantitatively hydrolyzed these conjugates within 3 min at room temperature directly on extraction columns. This on-column method saved time and eliminated the loss of valuable samples during transfer to the extraction column. LC–MS/MS-based calibration curves processed with this method showed good linearity, with r2 values exceeding 0.998. The intra- and inter-day accuracies and precisions of the method were 93.0–109.7% and 0.8–8.8%, respectively. The recovery efficiencies were in the range of 56.1–104.5%. Matrix effects were between 78.9 and 126.9%.ConclusionsWe have established an LC–MS/MS method for five psychoactive drugs in urine after enzymatic hydrolysis of glucuronide conjugates directly on extraction columns. The method was successfully applied to forensic autopsy samples. The established method will have broad applications, including forensic and clinical toxicological investigations.

Ortho-phenylphenol (OPP) has been widely used as a fungicide and preservative. Although low-dose studies have demonstrated its low toxicity in animals and humans, high-dose exposure to this contaminant has toxic effects that range from skin irritation to bladder cancer. Thus far, monitoring of OPP exposure in the general population has been performed by measuring OPP after urine hydrolysis with the β-glucuronidase/arylsulfatase enzyme and sometimes by the use of a mineral acid. We developed a sensitive, accurate, and robust method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to specifically measure two-phase II OPP metabolites excreted in human urine, OPP sulfate (OPP-S), and OPP glucuronide (OPP-G). Comparative analysis of urine samples from 50 volunteers living in the Quebec City area using a direct method and phosphoric acid hydrolysis method previously developed in our laboratory showed no statistically significant difference (p value for paired t test = 0.701) in OPP concentrations. Moreover, a significant difference showed that underestimation (p value for paired t test = 0.025) occurs when β-glucuronidase/arylsulfatase enzyme deconjugation is used. The LOD achieved by the direct method permits the detection of OPP-S and OPP-G metabolites in urine at the submicrogram per liter level. Graphical abstract ᅟ.

V-type nerve agents are among the most toxic organophosphorus chemical warfare agents, and they are under strict regulation and supervision by the OPCW (Organization for the Prohibition of Chemical Weapons). The V-type class of materials refers to a potentially large number of analogues and isomers. In order to expose instances of unfulfillment of the OPCW treaty, it is essential to have the ability to detect and identify "unknown" analogues of this family, even in the absence of an analytical standard. This work demonstrates a new automated tool for the detection and identification of V-type analogues, using high-resolution-accurate-mass LC-MS analysis, followed by "Compound Discoverer" software data processing. This software, originally developed for metabolism and metabolomics screening, is used here to automatically detect various V-type analogues by picking peaks and comparing them to "in-silico" calculated modifications made on a predefined basic V-backbone structure (according to the OPCW definitions for V-type agents). Subsequently, a complete structural elucidation for the proposed molecular formula is obtained by MS/MS data analysis of the suspected component, for both the V-type analogue (using ESI(+) analysis) as well as its hydrolysis product (using ESI(-) analysis) for a better elucidation of the phosphonate "head" structure. This method was found to be useful for the detection and identification of several "unknown" analogues, at low ng/mL levels in soil extracts.

R,S-Oxazepam and R,S-temazepam can be separated in their enantiomers by means of a chiral AGP column. The corresponding R- and S-glucuronide conjugates can be separated on a normal reversed-phase C18 column. Man conjugates the S-enantiomer of oxazepam and temazepam both better than the R-enantiomer. The urinary recovery of the glucuronides following either R,S,-oxazepam or R,S-temazepam almost amounts to 100% of the dose administered.

Iloperidone is a dopamine‐D2/serotonin‐5‐HT2antagonist used for the treatment of schizophrenia. The in vitro metabolism of iloperidone was conducted in human liver microsomes. With the addition of NADPH, methyl hydroxylation metabolite was the most abundant product and keto‐reduction metabolite andO‐demethylated metabolite were the major products. CYP2D6 and 3A4 were responsible for the hydroxylation and O‐demethylation, respectively. The reduction metabolite was mainly produced by cytosolic enzymes. Urine and plasma samples were collected after an oral administration of 4‐mg iloperidone in a clinical study. Two parallel metabolic processes were observed; one was carried out with the intact benzisoxazole and the other proceeded after the initial opening of the benzisoxazole ring. The reduction metabolite was the major component found in both plasma and urine of humans. The O‐demethylation metabolite was also found in plasma and urine and mainly existed in the form of glucuronide conjugate in urine.

Sulindac is a nonsteroidal anti‐inflammatory drug of the arylalkanoic acid class. It is used to reduce pain caused by arthritis, bursitis, gout, and tendinitis. The biotransformation of sulindac was investigated in rats, dogs, monkeys, and humans. The primary metabolic reactions were S‐oxidation of parent sulfoxide moiety to the corresponding sulfone and reversible reduction of sulfoxide to the sulfide. Sulindac sulfide is the pharmacologically active form of sulindac. Other metabolic reactions observed were the hydration of the parent double bond and methyl hydroxylation. Sulindac and its metabolites were also transformed as acyl glucuronide conjugates in urine.

High-performance liquid chromatography of imipramine and six metabolites in human plasma and urine.

An LC–MS method to determine concentrations of isoflavones and their sulfate and glucuronide conjugates in urine

Aniracetam is a pyrrolidinone derivative used as a cognition enhancer for the treatment of Alzheimer's disease and other neurodegenerative conditions. In vivo metabolic pathways of aniracetam in rats, dogs, and humans are shown. After oral and i.v. administrations of [ 14 C]‐aniracetam to human subjects, approximately 84, 0.8, and 11% of the radioactive dose was recovered in urine, feces, and expired air (as 14 CO 2 ), respectively, within 48 h. Approximately 70% of the dose was metabolized to 4‐ p ‐anisamidobutyric acid and excreted via urine. The remaining 30% of the dose was metabolized to 2‐pyrrolidinone and anisic acid. Further hydroxylation and oxidation of 2‐pyrrolidinone yielded 5‐hydroxy‐2‐pyrrolidinone and succinimide. Anisic acid was excreted mainly as glycine and glucuronide conjugates in urine.

Measurement of sulindac and its metabolites in human plasma and urine by high-performance liquid chromatography

The current studies examine acetaminophen pharmacokinetics and biotransformation in obese animals for possible shifts in metabolic conjugation reactions. Obesity was produced in Sprague-Dawley rats with an energy-dense cafeteria feeding regimen. Acetaminophen half-life remained unchanged and apparent volume of distribution increased slightly in obese versus pellet-fed control rats following an ip dose of 287 mg/kg. However, obese animals exhibited lower plasma concentrations of acetaminophen sulfate and excreted less sulfate conjugate but more glucuronide conjugate in urine. Absolute clearance of acetaminophen from plasma was similar for both groups of rats but formation clearance of acetaminophen sulfate was lower and formation clearance of acetaminophen glucuronide and was higher than control in obese rats. Renal clearance of unchanged drug and both conjugated metabolites appeared to rise with the degree of obesity. The many parallels in acetaminophen disposition shared with the obese human show the overfed rat to be a promising model for metabolic and physiologic changes associated with human obesity.

Context The therapeutic potential of andrographolide is hindered by its poor oral bioavailability and unpredictable pharmacokinetics, primarily due to its limited water solubility. Objective This work aimed to enhance the solubility and pharmacokinetics of andrographolide, a bioactive compound in Andrographis paniculata (Burm. f.) Nees (Acanthaceae), using solubilizing agents and a bioenhancer. Materials and methods Four groups of beagles were compared: (1) A. paniculata powder alone (control), (2) A. paniculata powder with 50% weight/weight (w/w) β-cyclodextrin solubilizer, (3) A. paniculata powder with 1% w/w sodium dodecyl sulfate (SDS) solubilizer, and (4) A. paniculata powder co-administered with 1% w/w SDS solubilizer and 10% piperine bioenhancer. All groups received a consistent oral dose of 3 mg/kg of andrographolide, administered both as a single dose and multiple doses over seven consecutive days. Results Thirteen chemical compounds were identified in A. paniculata powder, including 7 diterpenoids, 5 flavonoids, and 1 phenolic compound. A. paniculata co-administration with either 50% w/w β-cyclodextrin or 1% w/w SDS, alone or in combination with 10% w/w piperine, significantly increased systemic andrographolide exposure by enhancing bioavailability (131.01% to 196.05%) following single and multiple oral co-administration. Glucuronidation is one possible biotransformation pathway for andrographolide, as evidenced by the excretion of glucuronide conjugates in urine and feces. Conclusion The combination of solubilizing agents and a bioenhancer improved the oral bioavailability and pharmacokinetics of andrographolide, indicating potential implications for A. paniculata formulations and clinical therapeutic benefits. Further investigation in clinical studies is warranted.