Diagnosis of inborn errors of metabolism using filter paper urine,urease treatment,isotope dilution and gas chromatography–mass spectrometry

This review will be concerned primarily with a practical yet comprehensive diagnostic procedure for the diagnosis or even mass screening of a variety of metabolic disorders. This rapid, highly sensitive procedure offers possibilities for clinical chemistry laboratories to extend their diagnostic capacity to new areas of metabolic disorders. The diagnostic procedure consists of the use of urine or filter paper urine, preincubation of urine with urease, stable isotope dilution, and gas chromatography–mass spectrometry. Sample preparation from urine or filter paper urine, creatinine determination, stable isotope-labeled compounds used, and GC–MS measurement conditions are described. Not only organic acids or polar ones but also amino acids, sugars, polyols, purines, pyrimidines and other compounds are simultaneously analyzed and quantified. In this review, a pilot study for screening of 22 target diseases in newborns we are conducting in Japan is described. A neonate with presymptomatic propionic acidemia was detected among 10,000 neonates in the pilot study. The metabolic profiles of patients with ornithine carbamoyl transferase deficiency, fructose-1,6-bisphosphatase deficiency or succinic semialdehyde dehydrogenase deficiency obtained by this method are presented as examples. They were compared to those obtained by the conventional solvent extraction methods or by the tandem mass spectrometric method currently done with dried filter blood spots. The highly sensitive, specific and comprehensive features of our procedure are also demonstrated by its use in establishing the chemical diagnosis of pyrimidine degradation defects in order to prevent side effects of pyrimidine analogs such as 5-flurouracil, and the differential diagnosis of three types of homocystinuria, orotic aciduria, uraciluria and other urea cycle disorders. Evaluation of the effects of liver transplantation or nutritional conditions such as folate deficiency in patients with inborn errors of metabolism is also described.     

Screening for inborn errors of metabolism using gas chromatography–mass spectrometry

Screening of newborns for inborn errors of metabolism (IEM) in China is both a challenging and undeveloped area for gynecologists and pediatricians. Since 1999, the Capital Institute of Pediatrics has been studied as regards screening for IEM using advanced gas chromatography–mass spectrometry (GC–MS) method in collaboration with the Matsumoto Institute of Life Science (MILS), Japan, and has successfully diagnosed 51 cases of IEM in a total of 393 patients. Galactosemia, phenylketonuria and methylmalonic acidemia were the most frequent disorders among 51 cases of IEM. Treatment by suitable drugs and/or diet therapy was very effective in the most cases.     

Determination of bicyclol in dog plasma using liquid chromatography–tandem mass spectrometry

A sensitive and accurate method for determination of bicyclol in dog plasma was developed. Thermo Scientific TSQ Quantum triple quadrupole system with multiple ion monitoring (MIM) positive scanning mode was applied. Bicyclol and DDB (IS) sodium adduct molecular ions were monitored at m/z 413 and m/z 441 in both Q1 and Q3, respectively. The collision energy in Q2 was set to 15 eV. Precipitation method was employed in the extraction of bicyclol and DDB from the biological matrix. The method was validated over 1–500 ng/mL for bicyclol. The recovery was 96.5–109.5%, and the limit of quantitation (LOQ) detection was 1 ng/mL for bicyclol. The intra- and inter-day precision of the method at three concentrations was 3.3–14.3% with accuracy of 99.9–109.0%. The method was successfully applied to bioequivalence studies of bicyclol controlled-release formulation to obtain the pharmacokinetic parameters.     

Profiling degradants of paclitaxel using liquid chromatography–mass spectrometry and liquid chromatography–tandem mass spectrometry substructural techniques

A rapid and systematic strategy based on liquid chromatography–mass spectrometry (LC–MS) profiling and liquid chromatography–tandem mass spectrometry (LC–MS–MS) substructural techniques was utilized to elucidate the degradation products of paclitaxel, the active ingredient in Taxol. This strategy integrates, in a single instrumental approach, analytical HPLC, UV detection, full-scan electrospray MS, and MS–MS to rapidly and accurately elucidate structures of impurities and degradants. In these studies, degradants induced by acid, base, peroxide, and light were profiled using LC–MS and LC–MS–MS methodologies resulting in an LC–MS degradant database which includes information on molecular structures, chromatographic behavior, molecular mass, and MS–MS substructural information. The stressing conditions which may cause drug degradation are utilized to validate the analytical monitoring methods and serve as predictive tools for future formulation and packaging studies. Degradation products formed upon exposure to basic conditions included baccatin III, paclitaxel sidechain methyl ester, 10-deacetylpaclitaxel, and 7-epipaclitaxel. Degradation products formed upon exposure to acidic conditions included 10-deacetylpaclitaxel and the oxetane ring opened product. Treatment with hydrogen peroxide produced only 10-deacetylpaclitaxel. Exposure to high intensity light produced a number of degradants. The most abundant photodegradant of paclitaxel corresponded to an isomer which contains a C3–C11 bridge. These methodologies are applicable at any stage of the drug product cycle from discovery through development. This library of paclitaxel degradants provides a foundation for future development work regarding product monitoring, as well as use as a diagnostic tool for new degradation products.     

Quantitative analysis of lignocaine and metabolites in equine urine and plasma by liquid chromatography–tandem mass spectrometry

In this paper, a method for the sensitive and reproducible analysis of lignocaine and its four principal metabolites, monoethylxylidide (MEGX), glycylxylidide (GX), 3-hydroxylignocaine (3-HO-LIG), 4-hydroxylignocaine (4-HO-LIG) in equine urine and plasma samples is presented. The method uses liquid chromatography coupled to tandem mass spectrometry operating in electrospray ionisation positive ion mode (+ESI) via multiple reaction monitoring (MRM). Sample preparation involved solid-phase extraction using a mixed-mode phase. The internal standard adopted was lignocaine-d₁₀. Lignocaine and its metabolites were successfully resolved using an octadecylsilica reversed-phase column using a gradient mobile phase of acetonitrile and 0.1% (v/v) aqueous formic acid at a flow rate of 300 μL/min. Target analytes and the internal standard were determined by using the following transitions; lignocaine, 235.2 > 86.1; 3-HO-LIG and 4-HO-LIG, 251.2 > 86.1; MEGX, 207.1 > 58.1; GX, 179.1 > 122.1; and lignocaine-d₁₀, 245.2 > 96.1. Calibration curves were generated over the range 1–100 ng/mL for plasma samples and 1–1000 ng/mL for urine samples. The method was validated for instrument linearity, repeatability and detection limit (IDL), method linearity, repeatability, detection limit (MDL), quantitation limit (LOQ) and recovery. The method was successfully used to analyse both plasma and urine samples following a subcutaneous administration of lignocaine to a thoroughbred horse.     

Determination of ginsenoside Rg3 in human plasma and urine by high performance liquid chromatography–tandem mass spectrometry

Here we report a method capable of quantifying ginsenoside Rg3 in human plasma and urine. The method was validated over linear range of 2.5–1000.0 ng mL⁻¹ for plasma and 2.0–20.0 ng mL⁻¹ for urine using ginsenoside Rg1 as I.S. Compounds were extracted with ethyl acetate and analyzed by HPLC/MS/MS (API-4000 system equipped with ESI⁻ interface and a C₁₈ column). The inter- and intra-day precision and accuracy of QC samples were ≤8.5% relative error and were ≤14.4% relative standard deviation for plasma; were ≤5.6% and ≤13.3% for urine. The Rg3 was stable after 24 h at room temperature, 3 freeze/thaw cycles and 131 days at −30 °C. This method has been applied to pharmacokinetic study of ginsenoside Rg3 in human.     

Quantification of cyanuric acid residue in human urine using high performance liquid chromatography–tandem mass spectrometry

Concern has increased about the resulting health effects of exposure to melamine and its metabolic contaminant, cyanuric acid, after infants in China were fed baby formula milk products contaminated with these compounds. We have developed a selective and sensitive analytical method to quantify the amount of cyanuric acid in human urine. The sample preparation involved extracting free-form cyanuric acid in human urine using anion exchange solid phase extraction. Cyanuric acid was separated from its urinary matrix components on the polymeric strong anion exchange analytical column; the analysis was performed by high performance liquid chromatography–tandem mass spectrometry using negative mode electrospray ionization interface. Quantification was performed using isotope dilution calibration covering the concentration range of 1.00–200 ng/mL. The limit of detection was 0.60 ng/mL and the relative standard deviations were 2.8–10.5% across the calibration range. The relative recovery of cyanuric acid was 100–104%. Our method is suitable to detect urinary concentrations of cyanuric acid caused by either environmental exposures or emerging poisoning events.     

Determination of picamilon concentration in human plasma by liquid chromatography–tandem mass spectrometry

A rapid liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for the determination of picamilon concentration in human plasma. Picamilon was extracted from human plasma by protein precipitation. High performance liquid chromatography separation was performed on a Venusil ASB C₁₈ column with a mobile phase consisting of methanol ?10 mM ammonium acetate–formic acid (55:45:01, v/v/v) at a flow rate of 0.65 ml/min. Acquisition of mass spectrometric data was performed in selected reaction monitoring mode, using the transitions of m/z 209.0 → m/z (78.0 + 106.0) for picamilon and m/z 152.0 → m/z (93.0 + 110.0) for paracetamol (internal standard). The method was linear in the concentration range of 1.00–5000 ng/ml for the analyte. The lower limit of quantification was 1.00 ng/ml. The intra- and inter-assay precision were below 13.5%, and the accuracy was between 99.6% and 101.6%. The method was successfully applied to characterize the pharmacokinetic profiles of picamilon in healthy volunteers. This validated LC–MS/MS method was selective and rapid, and is suitable for the pharmacokinetic study of picamilon in humans.     

Determination of pyrrole–imidazole polyamide in rat plasma by liquid chromatography–tandem mass spectrometry

Pyrrole (Py)–imidazole (Im) polyamides synthesized by combining N-methylpyrrole and N-methylimidazole amino acids have been identified as novel candidates for gene therapy. In this study, a sensitive method using liquid chromatography–tandem mass spectrometry (LC–MS/MS) with an electrospray ionization (ESI) source was developed and validated for the determination and quantification of Py–Im polyamide in rat plasma. Py–Im polyamide was extracted from rat plasma by solid-phase extraction (SPE) using a Waters Oasis^® HLB cartridge. Separation was achieved on an ACQUITY UPLC HSS T3 (1.8 μm, 2.1 × 50 mm) column by gradient elution using acetonitrile:distilled water:acetic acid (5:95:0.1, v/v/v) and acetonitrile:distilled water:acetic acid (95:5:0.1, v/v/v). The method was validated over the range of 10–1000 ng/mL and the lower limit of quantification (LLOQ) was 10 ng/mL. This method was successfully applied to the investigation of the pharmacokinetics of Py–Im polyamide after intravenous administration.     

Quantification of levetiracetam in plasma of neonates by ultra performance liquid chromatography–tandem mass spectrometry

A sensitive and specific method using ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) was developed for the determination of levetiracetam (LEV) in plasma of neonates. A plasma aliquot of 50 μl was deproteinized by addition of 500 μl methanol which contained 5 μg/ml UCB 17025 as an internal standard. After centrifugation, 50 μl of supernatant was diluted with 1000 μl of 0.1% formic acid–10 mM ammonium formate in water (pH 3.5) (mobile phase solution A) and 2 μl was injected onto the UPLC-system. Compounds were separated on a Acquity UPLC BEH C₁₈ 2.1 mm × 100 mm column using gradient elution with mobile phase solution A and 0.1% formic acid in methanol (mobile phase solution B) with a flow rate of 0.4 ml/min and a total runtime of 4.0 min. LEV and the internal standard were detected using positive ion electrospray ionization followed by tandem mass spectrometry (ESI-MS/MS). The assay allowed quantification of LEV plasma concentrations in the range from 0.5 μg/ml to 150 μg/ml. Inter-assay inaccuracy was within ±2.7% and inter-assay precision was less than 4.5%. Matrix effects were minor: the recovery of LEV was between 97.7% and 100%. The developed method required minimal sample preparation and less plasma sample volume compared to earlier published LC–MS/MS methods. The method was successfully applied in a clinical pharmacokinetic study in which neonates received intravenous administrations of LEV for the treatment of neonatal seizures.     

Identification of YH439 and its metabolites in rat urine by gas chromatography–mass spectrometry and liquid chromatography–mass spectrometry

YH439 is a potential drug candidate for the treatment of various hepatic disorders. YH439 and its three metabolites have been identified in rat urine by liquid chromatography–mass spectrometry (LC–MS) and by gas chromatography (GC)–MS. Identification of YH439 and its metabolites was established by comparing their GC retention times and mass spectra with those of the synthesized authentic standards. Both electron impact- and positive chemical ionization MS have been evaluated. The metabolism study was performed in the rat using oral administration of the drug. A major metabolite (YH438) was identified as the N-dealkylation product of YH439. Other identified metabolites were caused by the loss of the methyl thiazolyl amine group (metabolite II) from YH439, the isopropyl hydrogen malonate group (metabolite IV) and the decarboxylated product (metabolite III) of metabolite II.     

Analysis of corticosteroids in equine urine by liquid chromatography–mass spectrometry

A liquid chromatography–mass spectrometry (LC–MS) method for the analysis of corticosteroids in equine urine was developed. Corticosteroid conjugates were hydrolysed with β-glucuronidase; free and enzyme-released corticosteroids were then extracted from the samples with ethyl acetate followed by a base wash. The isolated corticosteroids were detected by LC–MS and confirmed by LC–MS–MS in the positive atmospheric pressure chemical ionisation mode. Twenty-three corticosteroids (comprising hydrocortisone, deoxycorticosterone and 21 synthetic corticosteroids), each at 5 ng/ml in urine, could easily be analysed in 10 min.     

Simultaneous determination of paracetamol,pseudoephedrine, dextrophan and chlorpheniramine in human plasma by liquid chromatography–tandem mass spectrometry

For the first time, a highly sensitive and simple LC–MS/MS method after one-step precipitation was developed and validated for the simultaneous determination of paracetamol (PA), pseudoephedrine (PE), dextrophan (DT) and chlorpheniramine (CP) in human plasma using diphenhydramine as internal standard (IS). The analytes and IS were separated on a YMC-ODS-AQ C₁₈ Column (100 mm × 2.0 mm, 3 μm) by a gradient program with mobile phase consisting of 0.3% (v/v) acetic acid and methanol at a flow rate of 0.30 mL/min. Detection was performed on a triple quadrupole tandem mass spectrometer via electrospray ionization in the positive ion mode. The method was validated and linear over the concentration range of 10–5000 ng/mL for PA, 2–1000 ng/mL for PE, 0.05–25 ng/mL for DT and 0.1–50 ng/mL for CP. The accuracies as determined from quality control samples were in range of ?8.37% to 3.13% for all analytes. Intra-day and inter-day precision for all analytes were less than 11.54% and 14.35%, respectively. This validated method was successfully applied to a randomized, two-period cross-over bioequivalence study in 20 healthy Chinese volunteers receiving multicomponent formulations containing 325 mg of paracetamol, 30 mg of pseudoephedrine hydrochloride, 15 mg of dextromethorphan hydrobromide and 2 mg of chlorphenamine maleate.     

Analysis of cyclosporine A and its metabolites in rat urine and feces by liquid chromatography–tandem mass spectrometry

A sensitive and specific liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for the quantification of cyclosporine A (CyA) and the identification of its metabolites in rat urine and feces. The analytes were extracted from waste samples via liquid–liquid extraction. A Turboionspray source was used as a detector. It was operated in a positive ion mode with transitions of m/z 1225 → m/z 1112 for CyA and in a selected multiple reactions monitoring (MRM) mode with transitions of m/z 1239 → m/z 1099 for the internal standard (cyclosporine D, CyD). Linear calibration curves were obtained for CyA concentration ranges of 12.5–250 ng mL^?1 in urine and 2.5–375 ng mg^?1 in feces. The intra- and inter-day precision values (relative standard deviation) obtained were less than 8%, and the accuracy was within ±15% for each of the analytes. Extraction recoveries of CyA and CyD were both over 80%. The identification of the metabolites and elucidation of their structure were performed on the basis of their retention times and mass spectrometry fragmentation behaviors. A total of seven metabolites in rat feces were identified as dimethyl CyA, hydroxy CyA, and dihydroxy CyA after the oral administration of cyclosporine A-Eudragit^® S100 nanoparticles (CyA-NP). Six of these metabolites were also detected in rat urine. A possible metabolic pathway was also proposed. The newly developed method was proven to be sensitive, simple, reproducible, and suitable for the rapid determination of CyA. It was successfully employed to study the excretion of CyA in rats and could be used to better understand the in vivo metabolism of CyA-NP, a potentially effective nanoparticle system.     

Enantioselective determination of doxazosin in human plasma by liquid chromatography–tandem mass spectrometry using ovomucoid chiral stationary phase

An enantioselective and sensitive method was developed and validated for determination of doxazosin enantiomers in human plasma by liquid chromatography–tandem mass spectrometry. The enantiomers of doxazosin were extracted from plasma using ethyl ether/dichloromethane (3/2, v/v) under alkaline conditions. Baseline chiral separation was obtained within 9 min on an ovomucoid column using an isocratic mobile phase of methanol/5 mM ammonium acetate/formic acid (20/80/0.016, v/v/v) at a flow rate of 0.60 mL/min. Acquisition of mass spectrometric data was performed in multiple reaction monitoring mode, using the transitions of m/z 452 → 344 for doxazosin enantiomers, and m/z 384 → 247 for prazosin (internal standard). The method was linear in the concentration range of 0.100–50.0 ng/mL for each enantiomer using 200 μL of plasma. The lower limit of quantification (LLOQ) for each enantiomer was 0.100 ng/mL. The intra- and inter-assay precision was 5.0–11.1% and 5.7–7.6% for R-(−)-doxazosin and S-(+)-doxazosin, respectively. The accuracy was 97.4–99.5% for R-(−)-doxazosin and 96.8–102.8% for S-(+)-doxazosin. No chiral inversion was observed during the plasma storage, preparation and analysis. The method proved adequate for enantioselective pharmacokinetic studies of doxazosin after oral administration of therapeutic doses of racemic doxazosin.     

Improved liquid chromatography–tandem mass spectrometry method for the analysis of eptifibatide in human plasma

A rapid, selective and highly sensitive high performance liquid chromatography–tandem mass spectrometry method (LC–MS/MS) was developed and validated for the determination and pharmacokinetic investigation of eptifibatide in human plasma. Eptifibatide and the internal standard (IS), EPM-05, were extracted from plasma samples using solid phase extraction. Chromatographic separation was performed on a C₁₈ column at a flow rate of 0.5 mL/min. Detection of eptifibatide and the IS was achieved by tandem mass spectrometry with an electrospray ionization (ESI) interface in positive ion mode. Traditional multiple reaction monitoring (MRM) using the transition of m/z 832.6 → m/z 646.4 and m/z 931.6 → m/z 159.4 was performed to quantify eptifibatide and the IS, respectively. The calibration curves were linear over the range of 1–1000 ng/mL with the lower limit of quantitation validated at 1 ng/mL. The intra- and inter-day precisions were within 13.3%, while the accuracy was within ±7.6% of nominal values. The validated LC–MS/MS method was successfully applied for the evaluation of pharmacokinetic parameters of eptifibatide after intravenous (i.v.) administration of a 45 μg/kg bolus of eptifibatide to 8 healthy volunteers.     

Determination of eptifibatide concentration in human plasma utilizing the liquid chromatography–tandem mass spectrometry method

A sensitive and selective liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed to determine the concentration of eptifibatide in human plasma. Following protein precipitation, the analyte was separated on a reversed-phase C₁₈ column. Acetonitrile:5 mM ammonium acetate:acetic acid (30:70:0.1, v/v/v) was used at a flow-rate of 0.5 mL/min with the isocratic mobile phase. An API 4000 tandem mass spectrometer equipped with a Turbo IonSpray ionization source was used as the detector and was operated in the positive ion mode. “Truncated” multiple reaction monitoring using the transition of m/z 832.6 → m/z 832.6 and m/z 931.3 → m/z 931.3 was performed to quantify eptifibatide and the internal standard (EPM-05), respectively. The method had a lower limit of quantification of 4.61 ng/mL for eptifibatide. The calibration curve was demonstrated to be linear over the concentration range of 4.61 ? 2770 ng/mL. The intra- and inter-day precisions were less than 10.5% for each QC level, and the inter-day relative errors were 2.0%, 5.6%, and 2.8% for 9.22, 184, and 2490 ng/mL, respectively. The validated method was successfully applied to the quantification of eptifibatide concentration in human plasma after intravenous (i.v.) administration of a 270-μg/kg bolus of eptifibatide and i.v. administration of eptifibatide at a constant rate of infusion of 2 μg/(kg min) for 18 h in order to evaluate the pharmacokinetics.     

Determination of methocarbamol concentration in human plasma by high performance liquid chromatography–tandem mass spectrometry

A simple and reproducible high performance liquid chromatography–tandem mass spectrometric method was developed for methocarbamol analysis in human plasma. Methocarbamol and the internal standard (IS) were extracted by a protein precipitation method. Under isocratic separation condition the chromatographic run time was 3.0 min. The calibration curve was linear over a range of 150–12,000 ng/mL with good intraday assay and interday assay precision (CV% < 10.9%). The method was proven to be sensitive and selective for the analysis of methocarbamol in human plasma for bioequivalence study.     

Method for measurement of the quaternary amine compounds paraquat and diquat in human urine using high-performance liquid chromatography–tandem mass spectrometry

We have developed a highly selective and sensitive analytical method to quantify paraquat and diquat by use of high-performance liquid chromatography-tandem mass spectrometry (HPLC–MS/MS). The sample preparation includes solid phase extraction that uses weak cation exchange cartridges. These highly charged dual quaternary amines were not retained by standard reversed phase columns, but they could be adequately separated through HPLC with a HILIC column. The detection was carried out with a triple quadrupole mass spectrometer with an electrospray ionization probe in positive ion mode in multiple reaction monitoring. Repeated analysis in human urine samples spiked with low (5 ng/ml), medium (15 ng/ml), and high (30 ng/ml) concentrations of the analytes yielded relative standard deviations of less than 9%. The extraction efficiencies ranged from 77.7% to 94.2%. The limits of detection were in the range of 1 ng/ml.     

Determination of imidapril and imidaprilat in human plasma by high-performance liquid chromatography–electrospray ionization tandem mass spectrometry

A sensitive and specific assay of imidapril and its active metabolite, imidaprilat, in human plasma has been developed. This method is based on rapid isolation and high-performance liquid chromatography (HPLC)–electrospray ionization (ESI)-tandem mass spectrometry (MS–MS). Imidapril and imidaprilat were isolated from human plasma using OASIS HLB (solid-phase extraction cartridge), after deproteinization. The eluent from the cartridge was evaporated to dryness, and the residue was reconstituted in mobile phase and injected into the HPLC–ESI-MS–MS system. Each compound was separated on a semi-micro ODS column in acetonitrile–0.05% (v/v) formic acid (1:3, v/v). The selected ion monitoring using precursor→product ion combinations of m/z 406→234 and 378→206, was used for determination of imidapril and imidaprilat, respectively. The linearity was confirmed in the concentration range of 0.2 to 50 ng/ml in human plasma, and the precision of this assay, expressed as a relative standard deviation, was less than 13.2% over the entire concentration range with adequate assay accuracy. The HPLC–ESI-MS–MS method correlates well with the radioimmunoassay method, therefore, it is useful for the determination of imidapril and imidaprilat with sufficient sensitivity and specificity in clinical studies.