Comparison of three different chemiluminescence assays and a rapid liquid chromatography tandem mass spectrometry method for measuring serum aldosterone.

7-Ethyl-10-hydroxycamptothecin (SN-38) is an active metabolite of Irinotecan (CPT-11), an anticancer pro-drug. To support clinical pharmacokinetic studies for liposome based formulation of SN-38 (LE-SN38) in cancer patients, a rapid, simple and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the quantification of total SN-38 in human plasma. Sample preparation was carried out by one-step protein precipitation using cold acetonitrile with 0.5% acetic acid (v/v). Camptothecin was used as an internal standard (IS). Chromatographic separation of SN-38 and IS was achieved using a Synergi Hydro-RP column (C(18), 50 x 2 mm, 4 micro m), with a gradient elution of acetonitrile and 0.1% acetic acid. After ionization in electrospray source (positive ions), the acquisition was performed in the multiple reactions monitoring mode. Quantitation was accomplished using the precursor-->product ion combinations of m/z 393.1-->349.2 for SN-38 and 349.1-->305.1 for IS. The quantification limit of 0.05 ng/mL was achieved by using much lower volume (0.2 mL) of plasma and in the presence of LE-SN38. The method was validated over the concentration range of 0.05-400 ng/mL. Accuracy was within +/-12% of nominal at all concentration levels. Inter-day and intra-day precisions expressed as percentage coefficient of variation (%CVs) for quality control (QC) samples were less than 14 and 5%, respectively.

Background Measurement of serum dexamethasone during the overnight dexamethasone-suppression test has been recommended to reduce false-positive results when investigating Cushing's syndrome or increasingly commonly found adrenal incidentalomas. Despite this, there remains a paucity of well-validated dexamethasone methods currently available. Here, we describe the development of a rapid and sensitive liquid chromatography tandem mass spectrometry serum dexamethasone assay and verify its utility in a cohort of postmenopausal females. Method Isotopically labelled internal standard was added to samples prior to supported liquid extraction. Extracts were analysed using liquid chromatography tandem mass spectrometry in the positive electrospray ionization mode. Multiple reaction monitoring was used to detect dexamethasone and its corresponding internal standard transitions. Normal healthy postmenopausal women ( n = 95) were recruited and underwent an overnight dexamethasone suppression test, with serum dexamethasone and cortisol measurements at 09:00 after administration of oral dexamethasone 1 mg at 23:00 the night before. Results Mean intra- and inter-assay imprecision were 4.1% and 2.9%, respectively, for dexamethasone concentrations of 1.5, 6.0 and 12.0 nmol/L. Matrix effects were found to be negligible at 106-109% with recovery ranging from 96 to 100%. The limit of quantitation was 0.25 nmol/L, and structural analogue analysis proved the method to be robust against interferences. Applying a serum dexamethasone cut-off of >3.3 nmol/L was associated with a serum cortisol ≤50 nmol/L in 84/95 individuals. Conclusion We have developed a sensitive and robust liquid chromatography tandem mass spectrometry method for the quantitation of serum dexamethasone. The method can be used to identify false-positive results during the overnight dexamethasone suppression test or for pharmacokinetic studies.

Validation and comparison of a rapid liquid chromatography tandem mass spectrometry method for serum 25OHD with the efficiency of separating 3-epi 25OHD3

A rapid and sensitive high-performance liquid chromatography and electrospray tandem mass spectrometry method was developed and validated for estimation of fulvestrant in rabbit plasma using liquid-liquid extraction. The separation and quantification of fulvestrant were achieved by reverse-phase chromatography on a Sunfire C18 column (50 x 2.1. i.d., 3.5 microm) with isocratic elution at a flow rate of 300 microL/min using norethistrone as an internal standard from 500 microL plasma sample. The method was validated over the concentration range from 0.092 to 16.937 ng/mL with a lower limit of detection of 0.023 ng/mL. The intra-day and inter-day accuracy and precision were within 10%. The recovery was 85 and 90% for fulvestrant and norethistrone respectively. The chromatographic run time was only 2.5 min.

Comparison of plasma aldosterone measured by chemiluminescence immunoassay and liquid chromatography-tandem mass spectrometry in screening test for primary aldosteronism

Establishment of a rapid and simple liquid chromatography tandem mass spectrometry method for measuring aldosterone in urine

Automating DUID methods using robotics for rapid high throughput sample preparation – Analysis of Delta-9-Tetrahydrocannabinol and its major metabolite Delta-9-tetrahydrocannabinol acid in whole blood

A general liquid chromatography tandem mass spectrometry method for the quantitative determination of diquaternary ammonium gemini surfactant drug delivery agents in mouse keratinocytes’ cellular lysate

A simple, accurate and robust liquid chromatography tandem mass spectrometry (LC‐ESI‐MS/MS) method has been developed for estimation of nevirapine in human plasma. Abacavir sulphate was used as an internal standard (IS). The plasma filtrate obtained after solid phase extraction (SPE), using a hydrophilic‐lipophilic balance (HLB) cartridge, was submitted directly to short column liquid chromatography‐tandem mass spectrometric assay, with negligible matrix effect on the analysis. Turbo ion spray, operating in positive mode and multiple reaction monitoring (MRM) scan type was used to quantify nevirapine in human plasma. The extraction procedure yielded extremely clean extracts with a recovery of 91.85 and 97.71% for nevirapine and IS, respectively. The method is simple and reliable with a chromatographic run time of 2.5 min. The lower limit of quantification (LLOQ) found was 25 ng ml−1 with good accuracy and precision. The developed method was validated in the linear dynamic range of 25–5000 ng ml−1 with correlation coefficient (r)≥0.9996. The intra‐and inter‐batch precision for the samples at the LLOQ were 7.04 and 7.71%, respectively. The intra‐ and inter‐batch accuracy ranged from 91.17– 102.17%. The method was successfully applied for bioequivalence studies in 36 healthy Indian human subjects after oral administration of 10 mg ml−1 suspension formulations.

A sensitive, specific and rapid high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was described and validated for the quantification of ambroxol in human plasma using enalaprilat as the internal standard (IS). Chromatographic separation was performed on a Lichrospher CN column with a mobile phase of methanol and water (containing 0.1% formic acid) (70:30, v/v). The total run time was 5.0 min for each sample. The analytes was detected by mass spectrometry with electrospray ionization source in positive selected reaction monitoring mode. The precursor-fragment ion reaction for ambroxol was m/z 378.9 --> 263.8, and for IS was m/z 349.0 --> 205.9. The linearity was established over the concentration range of 1.56-400.00 ng/mL. The inter-day and the intra-day precisions were all within 10%. A simple protein precipitation with methanol was adopted for sample preparation. The extraction recoveries of ambroxol and IS were higher than 90.80%. The validated method was successfully applied in pharmacokinetic study after oral administration of 90 mg ambroxol to 24 healthy volunteers.

Liquid chromatography-tandem mass spectrometry-based recalibration reduces inter-platform variability in aldosterone detection across chemiluminescence immunoassay platforms.

Plasma aldosterone concentration has been usually measured by radioimmunoassay and chemiluminescent enzyme immunoassay which have demonstrated lack of high specificity and variability in assay performances. Liquid chromatography-tandem mass spectrometry is considered to be ideal measurement method of plasma aldosterone, however it takes relatively high cost and measurement time. We recently developed a novel non-competitive chemiluminescent enzyme immunoassay for plasma aldosterone concentrations by the fully automated assay system. We performed clinical validation of diagnostic ability of the new assay-based screening of 344 hypertensive patients including 133 patients with primary aldosterone producing adenoma, 100 patients with bilateral hyperaldosteronism, and 111 patients with essential hypertension. Passing-bablok analysis revealed that the new measurement of plasma aldosterone concentration was significantly correlated and almost equivalent to liquid chromatography-tandem mass spectrometry measurement of aldosterone; the regression slope and intercept were 0.962 and -0.043, and the correlation coefficient was 0.962, which is more similar than radioimmunoassay especially in lower range (< 10 ng/dL). Bland-Altman plot analysis with the mass-spectrometry measurement also demonstrated that both bias and limits of agreement with 95% confidence interval of the automated aldosterone assay were smaller than those of the radioimmunoassay, suggesting smaller systemic errors in the novel measurement. The lowest plasma aldosterone concentration and ratio of aldosterone-over-renin concentrations in aldosterone producing adenoma group were 6.0 ng/dL and 16.3 ng/dL per pg/mL/hr, respectively. We suggest if aldosterone-over-renin concentrations is over 15 ng/dL per pg/mL/hr, the patient should be performed confirmatory test for primary aldosteronism. The novel chemiluminescent enzyme immunoassay is clinically reliable alternative for conventional radioimmunoassay and liquid chromatography-tandem mass spectrometry to provide better throughput and cost-effectiveness in diagnosis of primary aldosteronism from larger number of hypertensive patients.

Objective To evaluate the consistency of different methods for detecting aldosterone concentration in blood and to establish a reference interval of serum aldosterone concentration by liquid chromatography-tandem mass spectrometry(LC-MS/MS). Methods Concentrations of blood aldosterone were measured by LC-MS/MS, chemiluminescent assays (Diasorin, Domestic A and B systems) and radioimmunoassay (RIA) in 138 healthy adults, 67 patients with essential hypertension and 23 patients with primary aldosteronism. Results Aldosterone concentrations measured by various methods were quiet different(P 0.05) between CLIA(Domestic B) and LC-MS/MS, 0.338(P<0.01) as well as between RIA and LC-MS/MS. Bland-Altman analysis for the measurements showed poor consistency among methods for aldosterone concentrations. The reference interval was 15.2-222.2 pg/ml for serum aldosterone concentrations by LC-MS/MS. Conclusions There are significant differences of blood aldosterone concentrations determined by different methods. Clinically, a specified reference interval might be needed while using different methods. Key words: Aldosterone; Liquid chromatography-tandem mass spectrometry; Chemiluminescent assays; Radioimmunoassay

Measurement of plasma aldosterone and renin concentration, or activity, is useful for selecting antihypertensive agents and detecting hyperaldosteronism in hypertensive patients. However, it takes several days to get results when measured by radioimmunoassay and development of more rapid assays has been long expected. We have developed chemiluminescent enzyme immunoassays enabling the simultaneous measurement of both aldosterone and renin concentrations in 10 minutes by a fully automated assay using antibody-immobilized magnetic particles with quick aggregation and dispersion. We performed clinical validation of diagnostic ability of this newly developed assay-based screening of 125 patients with primary aldosteronism from 97 patients with essential hypertension. Results of this novel assay significantly correlated with the results of radioimmunoassay (aldosterone, active renin concentration, and renin activity) and liquid chromatography-tandem mass spectrometry (aldosterone). The analytic sensitivity of this particularly novel active renin assay was 0.1 pg/mL, which was better than that of radioimmunoassay (2.0 pg/mL). The ratio of aldosterone-to-renin concentrations of 6.0 (ng/dL per pg/mL) provided 92.0% sensitivity and 76.3% specificity as a cutoff for differentiating primary aldosteronism from essential hypertension. This novel measurement is expected to be a clinically reliable alternative for conventional radioimmunoassay and to provide better throughput and cost effectiveness in diagnosis of hyperaldosteronism from larger numbers of hypertensive patients in clinical settings.

A 89-year-old woman was admitted to our hospital for syncope in 20XX-13. During the hospitalization, hypertension was pointed out. Despite 4 cm left adrenal mass suggestive of myelolipoma in contrast-enhanced magnetic resonance imaging (MRI), the serum and urine hormone levels in relation to secondary hypertension was within normal limits. Diagnosis of essential hypertension was made and treatment with olmesartan and azelnidipine was begun. In 20XX-5, her blood pressure was well controlled, and serum aldosterone level and active renin concentration (ARC) were 167 pg/ml and 6.8 pg/ml, respectively. Annual computed tomography (CT) scans showed virtually the same size of the adrenal tumor over 5 years. The trend toward increase in her blood pressure prompted us to repeat a blood test in 20XX, which turned out that serum aldosterone concentration was extremely high (1144 pg/ml), while ARC was suppressed to 1.7 pg/ml. In light of positive captopril challenge test, primary aldosteronism was suspected, and she was admitted to our hospital. However, systolic blood pressure was only mildly elevated (130 mmHg), though blood pressure medication had not been changed for 5 years. In addition, serum potassium value was 4.7 mEq/L and 24-hr urinary aldosterone was merely 3.4 μg/day under the salt-loaded state, which was inconsistent with exceedingly high serum aldosterone concentration and argued against the diagnosis of primary aldosteronism. Hence, validity of the method to quantitate serum aldosterone levels was questioned. To determine serum aldosterone concentration, radioimmunoassay (RIA) was applied in 20XX-5, yet chemiluminescent enzyme immunoassay (CLEIA) has been introduced since 20XX. Strikingly, reexamination of the same serum sample discovered that serum aldosterone value of 770 pg/ml in CLEIA was equivalent to 59 pg/ml by RIA method. As such, marked dissociation seems present between the two types of assays in this particular patient. Although tendency of correlation with regard to serum aldosterone levels between the two methods have been clarified, attention needs to be paid for the exception as in this case. Thus, it is fundamental to thoroughly evaluate patients with suspected primary aldosteronism as to whether the clinical course and serum aldosterone levels are compatible, and if not, it may be necessary to change the measurement method.