Measurement of serum aldosterone in picomolar level by LC-MS/MS using charge-tagged technique.

Aldosterone is a mineralocorticoid steroid hormone, the measurement of which in the clinical laboratory is principally performed for the investigation of primary hyperaldosteronism. Primary hyperaldosteronism is a specifically treatable and potentially curable form of hypertension, which typically presents as drug-resistant hypertension and, in up to 37% of cases, hypokalemia. Accurate measurement of aldosterone concentration is essential for correct diagnosis. The serum concentrations of aldosterone are in the picomolar range and therefore sensitive aldosterone assays are required. With the advancement in instrumentation of LC-MS/MS, the picomolar range of aldosterone can be easily measured by the newer models, but for those with a less sensitive instrument, special technique for sample preparation to enhance assay sensitivity is required. This work described the use of charge-tagging for the picomolar measurement of serum aldosterone in a less sensitive LC-MS/MS instrument. The assay was linear up to 3000 pmol/L with lower limit of quantitation at 80 pmol/L. The mean relative recovery was 96.5% with a range of 89.3-101.6% for aqueous calibrators and the mean relative recovery was 94.8% with a range of 87.5-101.4% for serum calibrators. Intra-assay CVs range from 8.2% to 11.3%, and inter-assay CVs ranged from 8.5% to 13.5% at concentration range from 229 to 1720 pmol/L. The LC-MS/MS method compared well (y = 1.04x + 8.97) with the in-use radioimmunoassay method. There was no significant difference found (p = 0.7135) between results determined by LC-MS/MS and radioimmunoassay method.

LC-MS/MS in the routine clinical laboratory: has its time come?

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been increasingly used in routine clinical laboratories during the last two decades. The high specificity, sensitivity, and multi-analyte potential make it an ideal alternative to immunoassays or conventional high-performance liquid chromatography (HPLC). It also provides higher throughput than gas chromatography-mass spectrometry (GC-MS). LC-MS/MS also offers higher flexibility than immunoassays because LC-MS/MS assays are typically developed in-house. In addition, abundant information can be obtained from a single LC-MS/MS run which can produce a large amount of quantitative or qualitative data. In this review, typical LC-MS/MS clinical applications are presented, personal experiences are shared, and strengths and weakness are discussed. It is foreseeable that LC-MS/MS will become a key instrument in routine clinical laboratories.

Determination of plasma cholesterol sulfate by LC-APCI-MS/MS in the context of pediatric autism.

Cholesterol sulfate (CS) has various biological functions. Previously, plasma CS was measured primarily as a means to diagnose X-linked ichthyosis; however, a recent hypothesis suggests that CS deficiency might be related to autism. As such, an assay capable of measuring both very high (in the case of X-linked ichthyosis) and very low (in the case of autism) plasma CS levels is required. Here we describe a novel LC-APCI-MS/MS method for the determination of CS in human plasma, and we propose normal CS ranges for children, based on studies of a local population of normal Chinese children between the ages of 2 and 10. In addition, we have used this method to measure plasma CS in autistic children. CS was isolated by solid-phase extraction, and quantified by isotope-dilution LC-APCI-MS/MS in negative ion mode monitoring 465.3>97.1 m/z (CS) and 472.3>97.1 m/z (CS-d7). Mean recovery of the assay ranged from 88.1 to 112.7%; within- and between-run imprecisions have CVs less than 7.2 and 8.1%, respectively. The assay was linear up to at least 100 µmol L(-1). The reference interval of plasma CS in males (range: 1.16-4.23 µmol L(-1)) was found to be higher than in females (range: 0.86-3.20 µmol L(-1)). Comparison of normal and autistic children showed no statistically significant difference in the plasma CS level. In conclusion, a robust LC-APCI-MS/MS method for plasma CS was developed, and a pediatric reference interval was derived from applying the method to normal and autistic children.

Combination of arsenic trioxide and chemotherapy in small cell lung cancer.

INTRODUCTION: Small cell lung cancer (SCLC) carries high mortality despite standard chemotherapy. Arsenic trioxide (ATO) has demonstrated clinical efficacy in leukemia and in vitro activity in various solid tumors. This study was conducted to determine the in vitro and in vivo combination effects of ATO and chemotherapy in SCLC. MATERIALS AND METHODS: The in vitro model consisted of 5 SCLC cell lines (H187, H526, H69, H841 and DMS79) and the anti-proliferative effects of ATO, cisplatin, etoposide or combinations thereof were measured. Synergism was determined by calculation of the combination index (CI) according to Chou and Talalay. Assays for apoptosis, intracellular glutathione (GSH) content, and mitochondrial membrane depolarization (MMD) were performed. Arsenic content was measured by inductively coupled plasma-mass spectrometry. Expression level of MRP1, MRP2 and pH2AX was detected by Western blot while cellular pH2AX level was monitored by immunofluorescent staining. An in vivo xenograft model in nude mice was established with a H841 cell line to test the effects of drug combinations. RESULTS: All 5 SCLC cell lines were sensitive to ATO, with IC(50) values (48 h) 1.6-8 µM. Synergistic or additive effects were obtained by combining cisplatin with ATO in all 5 cell lines. Combination of etoposide with ATO resulted in antagonistic or barely additive effects. Apoptotic assays and pH2AX immunofluorescent staining corroborated the synergistic combination of ATO and cisplatin. In addition, the ATO/cisplatin combination enhanced MMD, depleted GSH, downregulated MRP2 and elevated intracellular ATO content compared with either ATO or cisplatin alone. In vivo combination of ATO and cisplatin also demonstrated synergism in the H841 xenograft model. CONCLUSIONS: There was clinically relevant in vitro activity of ATO in a panel of 5 SCLC cell lines. Significant synergism was demonstrated with the ATO/cisplatin combination, while antagonism was noted with the ATO/etoposide combination in both in vitro and in vivo models.

Quantification of acylglycines in human urine by HPLC electrospray ionization-tandem mass spectrometry and the establishment of pediatric reference interval in local Chinese.

Urinary organic acids, plasma amino acids and acylcarnitine profile analyses are the main tools used to diagnose inborn errors of metabolisms (IEMs). However, without metabolic decompensation, these parameters are often not helpful. On the other hand, in cases of IEM, acylglycines are consistently raised even when patients appear to be in remission. This study aims to set-up a simple liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method for the determination of urine acylglycines, complementary to organic acid and acylcarnitine profiles, for the diagnosis of IEM. In addition, local reference intervals for various acylglycines are established by using this method. Acylglycines were isolated by solid-phase extraction, derivatized with n-butanol, separated by HPLC, and detected by ESI-MS/MS. Acylglycines were quantified with deuterated internal standards. Mean recoveries of acylglycines ranged from 90.2 to 109.3%. Within- and between-run imprecisions for all acylglycines have CVs less than 10%. Linear regression coefficients were greater than 0.99. Reference intervals were established according to CLSI guidelines by analyzing 204 samples from apparently healthy individuals less than 18 years of age. The distributions of AG in the "normal" urine were skewed towards the right. After log transformation, all the results were normally distributed. Partitioning into age group reference intervals was not indicated, according to the Harris and Boyd approach. In this context, a single reference interval for each acylglycine could be used. This method of urine acylglycines analysis is a powerful diagnostic tool, complementary to urine organic acids and plasma acylcarnitine profiling, for detecting certain inborn errors of metabolism.

Analytical challenges: determination of tetrodotoxin in human urine and plasma by LC-MS/MS.

Tetrodotoxin (TTX) is a powerful sodium channel blocker found in puffer fish and some marine animals. Cases of TTX poisoning most often result from puffer fish ingestion. Diagnosis is mainly from patient's signs and symptoms or the detection of TTX in the leftover food. If leftover food is unavailable, the determination of TTX in the patient's urine and/or plasma is essential to confirm the diagnosis. Although various methods for the determination of TTX have been published, most of them are for food tissue samples. Dealing with human urine and blood samples is much more challenging. Unlike in food, the amount of toxin in the urine and blood of a patient is generally extremely low; therefore a very sensitive method is required to detect it. In this regard, mass spectrometry (MS) methods are the best choice. Since TTX is a very polar compound, there will be lack of retention on conventional reverse-phase columns; use of ion pair reagent or hydrophilic interaction liquid chromatography (HILIC) can help solve this problem. The problem of ion suppression is another challenge in analyzing polar compound in biological samples. This review will discuss different MS methods and their pros and cons.

Metformin-associated lactic acidosis in Chinese patients with type II diabetes.

Metformin is a widely used antidiabetic agent that is generally considered safe. However, metformin-associated lactic acidosis (MALA), though not common, occurs from time to time and results in significantly high mortality. A series of 23 MALA cases in a local major hospital in Hong Kong is reported in this article to demonstrate the epidemiological data, risk factors, clinical features as well as the clinical outcomes for better understanding of this disease entity. It is the first MALA case series in which plasma metformin levels were assessed. However, the results show that plasma metformin levels in MALA bear no diagnostic and prognostic values. Risk factors of mortality were identified as shock and high plasma lactate levels. The majority of patients were found to have significantly raised creatinine versus a normal baseline value before the acute illness. Concomitant illnesses taking place alongside MALA were common. With a high utility rate of renal replacement therapy (82.6%) in the study group, the mortality rate was 30.4%.

Development and validation of a high-throughput double solid phase extraction-liquid chromatography-tandem mass spectrometry method for the determination of tetrodotoxin in human urine and plasma.

A sensitive analytical method for the determination of tetrodotoxin (TTX) in urine and plasma matrices was developed using double solid phase extraction (C18 and hydrophilic interaction liquid chromatography) and subsequent analysis by HPLC coupled with tandem mass spectrometry. The double SPE sample cleanup efficiently reduced matrix and ion suppression effects. Together with the use of ion pair reagent in the mobile phase, isocratic elution became possible which enabled a shorter analysis time of 5.5 min per sample. The assay results were linear up to 500 ng mL(-1) for urine and 20 ng mL(-1) for plasma. The limit of detection and limit of quantification were 0.13 ng mL(-1) and 2.5 ng mL(-1), respectively, for both biological matrices. Recoveries were in the range of 75-81%. To eliminate the effect of dehydration and variations in urinary output, urinary creatinine-adjustment was made. TTX was quantified in eight urine samples and seven plasma samples from eight patients suspected of having TTX poisoning. TTX was detected in all urine samples, with concentrations ranging from 17.6 to 460.5 ng mL(-1), but was not detected in any of the plasma samples. The creatinine-adjusted TTX concentration in urine (ranging from 7.4 to 41.1 ng µmol(-1) creatinine) correlated well with the degree of poisoning as observed from clinical symptoms.

Improved liquid chromatography-tandem mass spectrometry method in clinical utility for the diagnosis of Cushing's syndrome.

Determination of urinary free cortisol is one of the first lines in screening for the diagnosis of Cushing's syndrome where its measurement is mostly done by immunoassay. Although easy to perform, immunoassays suffer from the problem of assay interferences and are unable to measure cortisone levels. To enhance such techniques for clinical diagnosis, an improved liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed for the simultaneous determination of urinary free cortisol and cortisone. The leftover urine samples from immunoassay were collected and subjected to facile solid-phase extraction cleanup. In the analysis of 130 urine samples from patients, 65 (50%) were found to have elevated urinary free cortisol (UFC) by immunoassay; but only 13 (10.8%) were found to have elevated UFC by this improved LC-MS/MS method. Nine out of the 13 patients, which showed elevated UFC by LC-MS/MS, were surgically confirmed to have Cushing's syndrome/disease. By setting a two times upper limit as a cut-off, the immunoassay gave a positive predictive value of 43.5%, whilst by using the improved method, a positive predictive value of 90% was obtained. Although several tests have been used extensively in first line screening for the diagnosis of Cushing's syndrome, none has ever shown with full capability of distinguishing all cases of Cushing's syndrome from normal and/or obese individuals. This method has shown superior analytical advantages over existing immunoassay type in terms of sensitivity, specificity and capability to diagnose Cushing's syndrome. Comparison between existing spectrometric methods, the reported developed method shown here, provides a simpler sample preparation procedure and meets with the high throughput demand of clinical laboratories.

Determination of mercury in whole blood and urine by inductively coupled plasma mass spectrometry.

The conventional method for the determination of mercury in clinical samples is cold vapor atomic absorption spectrometry. Sample digestion or pretreatment require large sample volume and long sample preparation time. The inductively coupled plasma mass spectrometry (ICP-MS) method developed in this study requires only 100 microL of sample with practically no preparation, except for dilution with diluent. Significant savings in sample volumes, reagents, technician time, and analysis time are realized. Among different types of diluents, the one containing acid, tert-butanol, and potassium dichromate gave the best results to remove the mercury memory effect. The interassay precisions for whole blood and urine were < 5% and < 8%, respectively, and the intra-assay precisions were < 3% and < 7%, respectively. The lower limits of detection were 0.13, 0.17, and 0.26 microg/L for aqueous standard, urine, and whole blood, respectively. The developed ICP-MS method correlated well with the atomic absorption method and can offer an alternative to the atomic absorption method for mercury analysis with less sample volume requirement as well as shorter analysis time.