HPLC-electrospray Tandem Mass Spectrometry Research Articles

Analysis of free and conjugated varanic acid diastereomers using HPLC electrospray tandem-mass spectrometry in patients with D-bifunctional protein deficiency

Analysis of free and conjugated varanic acid diastereomers using HPLC electrospray tandem-mass spectrometry in patients with D-bifunctional protein deficiency

Defects in Pyrimidine Degradation Identified by HPLC-Electrospray Tandem Mass Spectrometry of Urine Specimens or Urine-soaked Filter Paper Strips

Urinary concentrations of thymine, uracil, and their degradation products are useful indicators of deficiencies of enzymes of the pyrimidine degradation pathway. We describe a rapid, specific method to measure these concentrations to detect inborn errors of pyrimidine metabolism. We used urine or urine-soaked filter-paper strips as samples and measured thymine, uracil, and their degradation products dihydrothymine, dihydrouracil, N:-carbamyl-ss-aminoisobutyric acid, and N:-carbamyl-ss-alanine. Reversed-phase HPLC was combined with electrospray ionization tandem mass spectrometry, and detection was performed by multiple-reaction monitoring. Stable-isotope-labeled reference compounds were used as internal standards. All pyrimidine degradation products could be measured in one analytical run of 15 min. Detection limits were 0.4-4 micromol/L. The intraassay imprecision (CV) of urine samples with added compounds was 1.3-12% for liquid urines and 1. 0-10% for filter-paper extracts of the urines. The interassay imprecision (CV) was 3-11% (100-200 micromol/L). Recoveries were 89-99% at 100-200 micromol/L and 95-106% at 1 mmol/L in liquid urines, and 93-103% at 100-200 micromol/L and 100-106% at 1 mmol/L in filter-paper samples. Correct identifications of deficiencies of the pyrimidine-degrading enzymes were readily made with urine samples from patients with known defects. HPLC with electrospray ionization tandem mass spectrometry allows rapid testing for disorders of the pyrimidine degradation pathway, and filter-paper samples allow easy collection, transport, and storage of urine samples.

Rapid Screening of High-Risk Patients for Disorders of Purine and Pyrimidine Metabolism Using HPLC-Electrospray Tandem Mass Spectrometry of Liquid Urine or Urine-soaked Filter Paper Strips

A rapid and specific screening method for patients at risk of inherited disorders of purine and pyrimidine metabolism is desirable because symptoms are varied and nonspecific. The aim of this study was to develop a rapid and specific method for screening with use of liquid urine samples or urine-soaked filter paper strips. Reverse-phase HPLC was combined with electrospray ionization (ESI), tandem mass spectrometry (MS/MS), and detection performed by multiple reaction monitoring. Transitions and instrument settings were established for 17 purines or pyrimidines. Stable-isotope-labeled reference compounds were used as internal standards when available. Total analysis time of this method was 15 min, approximately one-third that of conventional HPLC with ultraviolet detection. Recoveries were 96-107% in urine with added analyte, with two exceptions (hypoxanthine, 64%; xanthine, 79%), and 89-110% in urine-soaked filter paper strips, with three exceptions (hypoxanthine, 65%; xanthine, 77%; 5-hydroxymethyluracil, 80%). The expected abnormalities were easily found in samples from patients with purine nucleoside phosphorylase deficiency, ornithine transcarbamylase deficiency, molybdenum cofactor deficiency, adenylosuccinase deficiency, or dihydropyrimidine dehydrogenase deficiency. HPLC-ESI MS/MS of urine allows rapid screening for disorders of purine and pyrimidine metabolism. The filter paper strips offer the advantage of easy collection, transport, and storage of the urine samples.

Rapid analysis of conjugated bile acids in plasma using electrospray tandem mass spectrometry: application for selective screening of peroxisomal disorders.

The final step in bile acid biosynthesis takes place in peroxisomes and involves oxidative cleavage of the side-chain of C 27 -β-cholestanoic acids, leading to the formation of the primary bile acids cholic and chenodeoxycholic acid. Therefore, in a number of peroxisomal disorders, including both peroxisomal biogenesis defects and isolated defects of peroxisomal β-oxidation, the accumulation of serum C 27 -5β-cholestanoic acids can be observed (Bjorkhem 1994; Clayton et al 1987; Libert et al 1991; Wanders et al 1995). Since routine gas chromatography mass-spectrometry (GC-MS) analysis of plasma bile acids requires laborious sample preparation including enzymatic and/or chemical deconjugation of plasma bile acids prior to derivatization and GC-MS analysis, we have developed a rapid HPLC electrospray tandem mass spectrometry (ESI-MS/MS) method for analysing taurine- and glycine-conjugated bile acids.

Quantitative analysis of sirolimus (Rapamycin) in blood by high-performance liquid chromatography–electrospray tandem mass spectrometry

We report here a quantitative method for the analysis of sirolimus in blood using solid-phase sample preparation and HPLC–electrospray-tandem mass spectrometry detection. Blood samples (500 μl) were prepared by pre-treatment with acetonitrile: 15 m M zinc sulphate (70:30, v/v), containing 32-demethoxysirolimus (internal standard) and C 18 solid-phase extraction. The electrospray conditions were chosen to enhance the [M+NH 4] + species at the expense of other species. Detection was by multiple reactant monitoring with the mass transitions m/z 931.8→864.6 and m/z 901.8→834.4 employed for sirolimus and the internal standard, respectively. The method was linear over the range 0.2 to 100.0 μg l −1. The accuracy and inter-day precision, over this concentration range, was 94.4% to 104.4% and 1.4% to 5.0%, respectively. The accuracy and total precision at the limit of quantitation (0.2 μg l −1) was 103.0% and 10.8%, respectively. The mean absolute recovery of sirolimus and the internal standard were 80.5% and 81.3%, respectively. The sensitivity and analytical concentration range of the method make it suitable for therapeutic drug monitoring and pharmacokinetic studies. Further, the ability of the method to measure parent drug specifically will facilitate the evaluation of immunoassays for sirolimus.

Endogenous benzodiazepine-like compounds and diazepam binding inhibitor in serum of patients with liver cirrhosis with and without overt encephalopathy

Background/Aim—Despite some controversy, it has been suggested that endogenous benzodiazepine plays a role in the pathogenesis of hepatic encephalopathy. The aim of the present study was to evaluate the concentrations...

Microscale high-performance liquid chromatography–electrospray tandem mass spectrometry assay for cyclosporin A in blood

To facilitate quantitative analysis of cyclosporin A in low volume blood samples we developed a sensitive and specific microscale reversed-phase HPLC–electrospray tandem mass spectrometry assay. Blood samples (100 μl) were prepared by acetonitrile precipitation and C 18 solid-phase extraction. Detection was by multiple-reactant monitoring. The method was linear over the range 5–1000 μg/l ( r≥0.997) with accuracy between 95.4 and 102.0% over this range. Total imprecision was 11.1% at 10 μg/l and 2.8% at 800 μg/l. Absolute recovery of cyclosporin A and internal standard was 72.5 and 73.3%, respectively. When this method was evaluated against a conventional HPLC with UV detection, in patient samples, they were interchangeable ( y=0.988 x+10.0, r=0.996). This HPLC–ESI-MS–MS method will be applicable to therapeutic monitoring in paediatric transplant patients and multiple point pharmacokinetic studies in animals and humans.