Quantitative acylcarnitine determination by UHPLC-MS/MS--Going beyond tandem MS acylcarnitine "profiles".

Tandem MS "profiling" of acylcarnitines and amino acids was conceived as a first-tier screening method, and its application to expanded newborn screening has been enormously successful. However, unlike amino acid screening (which uses amino acid analysis as its second-tier validation of screening results), acylcarnitine "profiling" also assumed the role of second-tier validation, due to the lack of a generally accepted second-tier acylcarnitine determination method. In this report, we present results from the application of our validated UHPLC-MS/MS second-tier method for the quantification of total carnitine, free carnitine, butyrobetaine, and acylcarnitines to patient samples with known diagnoses: malonic acidemia, short-chain acyl-CoA dehydrogenase deficiency (SCADD) or isobutyryl-CoA dehydrogenase deficiency (IBD), 3-methyl-crotonyl carboxylase deficiency (3-MCC) or ß-ketothiolase deficiency (BKT), and methylmalonic acidemia (MMA). We demonstrate the assay's ability to separate constitutional isomers and diastereomeric acylcarnitines and generate values with a high level of accuracy and precision. These capabilities are unavailable when using tandem MS "profiles". We also show examples of research interest, where separation of acylcarnitine species and accurate and precise acylcarnitine quantification is necessary.

Ammonia control in children ages 2 months through 5 years with urea cycle disorders: comparison of sodium phenylbutyrate and glycerol phenylbutyrate.

OBJECTIVES: To examine ammonia levels, pharmacokinetics, and safety of glycerol phenylbutyrate (GPB; also referred to as HPN-100) and sodium phenylbutyrate (NaPBA) in young children with urea cycle disorders (UCDs). STUDY DESIGN: This open label switch-over study enrolled patients ages 29 days to under 6 years taking NaPBA. Patients underwent 24-hour blood and urine sampling on NaPBA and again on a phenylbutyric acid-equimolar dose of GPB and completed questionnaires regarding signs and symptoms associated with NaPBA and/or their UCD. RESULTS: Fifteen patients (8 argininosuccinate lyase deficiency, 3 argininosuccinic acid synthetase deficiency, 3 ornithine transcarbamylase deficiency, 1 arginase deficiency) ages 2 months through 5 years enrolled in and completed the study. Daily ammonia exposure (24-hour area under the curve) was lower on GPB and met predefined noninferiority criteria (ratio of means 0.79; 95% CI 0.593-1.055; P=.03 Wilcoxon; 0.07 t test). Six patients experienced mild adverse events on GPB; there were no serious adverse events or significant laboratory changes. Liver tests and argininosuccinic acid levels among patients with argininosuccinate lyase deficiency were unchanged or improved on GPB. Eleven of 15 patients reported 35 symptoms on day 1; 23 of these 35 symptoms improved or resolved on GPB. Mean systemic exposure to phenylbutyric acid, phenylacetic acid, and phenylacetylglutamine (PAGN) were similar and phenylacetic acid exposure tended to be higher in the youngest children on both drugs. Urinary PAGN concentration was greater on morning voids and varied less over 24 hours on GPB versus NaPBA. CONCLUSIONS: GPB results in more evenly distributed urinary output of PAGN over 24 hours were associated with fewer symptoms and offers ammonia control comparable with that observed with NaPBA in young children with UCDs.

Urinary phenylacetylglutamine as dosing biomarker for patients with urea cycle disorders.

UNLABELLED: We have analyzed pharmacokinetic data for glycerol phenylbutyrate (also GT4P or HPN-100) and sodium phenylbutyrate with respect to possible dosing biomarkers in patients with urea cycle disorders (UCD). STUDY DESIGN: These analyses are based on over 3000 urine and plasma data points from 54 adult and 11 pediatric UCD patients (ages 6-17) who participated in three clinical studies comparing ammonia control and pharmacokinetics during steady state treatment with glycerol phenylbutyrate or sodium phenylbutyrate. All patients received phenylbutyric acid equivalent doses of glycerol phenylbutyrate or sodium phenylbutyrate in a cross over fashion and underwent 24-hour blood samples and urine sampling for phenylbutyric acid, phenylacetic acid and phenylacetylglutamine. RESULTS: Patients received phenylbutyric acid equivalent doses of glycerol phenylbutyrate ranging from 1.5 to 31.8 g/day and of sodium phenylbutyrate ranging from 1.3 to 31.7 g/day. Plasma metabolite levels varied widely, with average fluctuation indices ranging from 1979% to 5690% for phenylbutyric acid, 843% to 3931% for phenylacetic acid, and 881% to 1434% for phenylacetylglutamine. Mean percent recovery of phenylbutyric acid as urinary phenylacetylglutamine was 66.4 and 69.0 for pediatric patients and 68.7 and 71.4 for adult patients on glycerol phenylbutyrate and sodium phenylbutyrate, respectively. The correlation with dose was strongest for urinary phenylacetylglutamine excretion, either as morning spot urine (r = 0.730, p < 0.001) or as total 24-hour excretion (r = 0.791 p<0.001), followed by plasma phenylacetylglutamine AUC(24-hour), plasma phenylacetic acid AUC(24-hour) and phenylbutyric acid AUC(24-hour). Plasma phenylacetic acid levels in adult and pediatric patients did not show a consistent relationship with either urinary phenylacetylglutamine or ammonia control. CONCLUSION: The findings are collectively consistent with substantial yet variable pre-systemic (1st pass) conversion of phenylbutyric acid to phenylacetic acid and/or phenylacetylglutamine. The variability of blood metabolite levels during the day, their weaker correlation with dose, the need for multiple blood samples to capture trough and peak, and the inconsistency between phenylacetic acid and urinary phenylacetylglutamine as a marker of waste nitrogen scavenging limit the utility of plasma levels for therapeutic monitoring. By contrast, 24-hour urinary phenylacetylglutamine and morning spot urine phenylacetylglutamine correlate strongly with dose and appear to be clinically useful non-invasive biomarkers for compliance and therapeutic monitoring.

Urinary 2-hydroxy-5-oxoproline, the lactam form of α-ketoglutaramate, is markedly increased in urea cycle disorders.

α-Ketoglutaramate (KGM) is the α-keto acid analogue of glutamine, which exists mostly in equilibrium with a lactam form (2-hydroxy-5-oxoproline) under physiological conditions. KGM was identified in human urine and its concentration quantified by gas chromatography/mass spectrometry (GC/MS). The keto acid was shown to be markedly elevated in urine obtained from patients with primary hyperammonemia due to an inherited metabolic defect in any one of the five enzymes of the urea cycle. Increased urinary KGM was also noted in other patients with primary hyperammonemia, including three patients with a defect resulting in lysinuric protein intolerance and one of two patients with a defect in the ornithine transporter I. These findings indicate disturbances in nitrogen metabolism, most probably at the level of glutamine metabolism in primary hyperammonemia diseases. Urinary KGM levels, however, were not well correlated with secondary hyperammonemia in patients with propionic acidemia or methylmalonic acidemia, possibly as a result, in part, of decreased glutamine levels. In conclusion, the GC/MS procedure has the required lower limit of quantification for analysis of urinary KGM, which is markedly increased in urea cycle disorders and other primary hyperammonemic diseases.

Simultaneous LC-MS/MS determination of phenylbutyrate, phenylacetate benzoate and their corresponding metabolites phenylacetylglutamine and hippurate in blood and urine.

Inborn errors of urea metabolism result in hyperammonemia. Treatment of urea cycle disorders can effectively lower plasma ammonium levels and results in survival in the majority of patients. Available medications for treating urea cycle disorders include sodium benzoate (BA), sodium phenylacetate (PAA), and sodium phenylbutyrate (PBA) and are given to provide alternate routes for disposition of waste nitrogen excretion. In this study, we develop and validate a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of benzoic acid, phenylacetic acid, phenylbutyric acid, phenylacetylglutamine, and hippuric acid in plasma and urine from children with inborn errors of urea synthesis. Plasma extracts and diluted urine samples were injected on a reverse-phase column and identified and quantified by selected reaction monitoring (SRM) in negative ion mode. Deuterated analogues served as internal standards. Analysis time was 7 min. Assay precision, accuracy, and linearity and sample stability were determined using enriched samples. Quantification limits of the method were 100 ng/ml (0.3-0.8 µmol/L) for all analytes, and recoveries were >90%. Inter- and intraday relative standard deviations were <10%. Our newly developed LC-MS/MS represents a robust, sensitive, and rapid method that allows simultaneous determination of the five compounds in plasma and urine.

Quantification of urinary derivatives of Phenylbutyric and Benzoic acids by LC-MS/MS as treatment compliance biomarkers in Urea Cycle disorders.

PURPOSE: Salts of phenylacetic acid (PAA) and phenylbutyric acid (PBA) have been used for nitrogen elimination as a treatment for hyperammonaemia caused by urea cycle disorders (UCD). A new analytical method for PBA measurement in urine which helps to evaluate the drug adherence has been implemented. METHODS: Urine specimens from UCD patients receiving PBA were analysed by tandem mass spectrometry to measure urine phenylacetylglutamine (PAGln). Some clinical and biochemical data for each patient were collected. RESULTS: Our study included 87 samples from 40 UCD patients. The PAGln levels did not correlate with height, weight or age. However, the PAGln values showed correlation with PBA dose (r = 0.383, P = 0.015). Plasma glutamine and ammonia levels presented a positive correlation (r = 0.537, P < 0.001). The stability for PAGln in urine was determined at different storage temperatures. CONCLUSIONS: We have developed a simple method for the determination of PAGln in urine, which acts as useful biomarker of effective drug delivery. PAGln in urine is stable at room temperature at least for 15 days, and for several months when frozen at -20 °C. This procedure is useful for the optimization and monitorization of the drug dose allowing the use of spot urine samples.

High-throughput tandem mass spectrometry multiplex analysis for newborn urinary screening of creatine synthesis and transport disorders, Triple H syndrome and OTC deficiency.

BACKGROUND: Creatine synthesis and transport disorders, Triple H syndrome and ornithine transcarbamylase deficiency are treatable inborn errors of metabolism. Early screening of patients was found to be beneficial. Mass spectrometry analysis of specific urinary biomarkers might lead to early detection and treatment in the neonatal period. We developed a high-throughput mass spectrometry methodology applicable to newborn screening using dried urine on filter paper for these aforementioned diseases. METHODS: A high-throughput methodology was devised for the simultaneous analysis of creatine, guanidineacetic acid, orotic acid, uracil, creatinine and respective internal standards, using both positive and negative electrospray ionization modes, depending on the compound. RESULTS: The precision and accuracy varied by <15%. Stability during storage at different temperatures was confirmed for three weeks. The limits of detection and quantification for each biomarker varied from 0.3 to 6.3 µmol/l and from 1.0 to 20.9 µmol/l, respectively. Analyses of urine specimens from affected patients revealed abnormal results. Targeted biomarkers in urine were detected in the first weeks of life. CONCLUSIONS: This rapid, simple and robust liquid chromatography/tandem mass spectrometry methodology is an efficient tool applicable to urine screening for inherited disorders by biochemical laboratories.

Population pharmacokinetic modeling and dosing simulations of nitrogen-scavenging compounds: disposition of glycerol phenylbutyrate and sodium phenylbutyrate in adult and pediatric patients with urea cycle disorders.

Sodium phenylbutyrate and glycerol phenylbutyrate mediate waste nitrogen excretion in the form of urinary phenylacetylglutamine (PAGN) in patients with urea cycle disorders (UCDs); rare genetic disorders characterized by impaired urea synthesis and hyperammonemia. Sodium phenylbutyrate is approved for UCD treatment; the development of glycerol phenylbutyrate afforded the opportunity to characterize the pharmacokinetics (PK) of both compounds. A population PK model was developed using data from four Phase II/III trials that collectively enrolled patients ages 2 months to 72 years. Dose simulations were performed with particular attention to phenylacetic acid (PAA), which has been associated with adverse events in non-UCD populations. The final model described metabolite levels in plasma and urine for both drugs and was characterized by (a) partial presystemic metabolism of phenylbutyric acid (PBA) to PAA and/or PAGN, (b) slower PBA absorption and greater presystemic conversion with glycerol phenylbutyrate, (c) similar systemic disposition with saturable conversion of PAA to PAGN for both drugs, and (d) body surface area (BSA) as a significant covariate accounting for age-related PK differences. Dose simulations demonstrated similar PAA exposure following mole-equivalent PBA dosing of both drugs and greater PAA exposure in younger patients based on BSA.

Quantitation of orotic acid in urine using isotope dilution-selected ion gas chromatography-mass spectrometry.

The measurement of urinary orotic acid excretion is an important test for establishing a diagnosis of hereditary orotic aciduria, a genetic defect of pyrimidine biosynthesis. Measurement of secondary urinary orotic acid elevation is also an important clinical test for the differential diagnosis of hyperammonemia due to some of the primary disorders of the urea cycle including ornithine transcarbamylase (OTC) deficiency, and the hyperornithinemia-hyperammonemia-homocitrullinemia (HHH) syndrome. Low levels of orotic acid are observed in carbamylphosphate synthetase (CPS) defects. This method utilizes a stable-isotope labeled internal standard (1, 3-(15)N-orotic acid), which is added to the standards, controls, and patient samples prior to extraction. Interference from urea is removed by incubation of samples with urease and the orotic acid is derivatized by trimethylsilylation. Quantitation is made against an eight-point standard curve using specific selected ions from both the labeled and unlabeled orotic acid.