Preparation of novel β-cyclodextrin functionalized monolith and its application in chiral separation

A novel β-cyclodextrin (β-CD) functionalized organic polymer monolith was prepared by covalently bonding ethylenediamine-β-CD (EDA-β-CD) to poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) (poly(GMA-co-EGDMA)) monolith via ring opening reaction of epoxy groups. SEM characterization was performed to confirm the homogeneity of the monolithic polymer. The resulting monolith was then characterized by DSC and XPS elemental analysis to study the thermal stability of the monolith, and to prove the successful immobilization of β-CD on the polymer substrate. The β-CD ligand density of 0.68 mmol g^?1 was obtained for the modified monolith, indicating the high reactivity and efficiency of the EDA-β-CD modifier. The ethylenediamine-β-CD functionalized monoliths were used for the chiral separation of ibuprofen racemic mixture and showed promising results.     

Elimination of diastereomer interference to determine Telcagepant (MK-0974) in human plasma using on-line turbulent-flow technology and off-line solid-phase extraction coupled with liquid chromatography/tandem mass spectrometry

To eliminate the diastereomer interference on Telcagepant (MK-0974) determination during clinical study support, on-line high turbulent-flow liquid chromatography (HTLC) methods, HTLC-A and HTLC-B that covered dynamic range of 0.5–500 nM and 5–5000 nM, respectively, were developed. To meet the requirement of rapid assay transfer among multiple laboratories and analysts, a solid-phase extraction (SPE) assay was derived from the existing HTLC-B assay under the same dynamic range. The on-line HTLC assays were achieved through direct injection of plasma samples, extraction of analyte with a Cohesive C₁₈ column (50 mm × 0.5 mm, 50 μm), followed by HPLC separation on a FluoPhase RP column (100 mm × 2.1 mm, 5 μm) and MS/MS detection. The off-line SPE assay used Waters Oasis^®HLB μElution plate to extract the analytes from plasma matrix before injecting on a FluoPhase RP column (150 mm × 2.1 mm, 5 μm) for LC–MS/MS analysis. Under both on-line and off-line assay conditions, the diastereomer 1c was chromatographically separated from MK-0974. Cross-validation with the pooled samples demonstrated that both on-line and off-line assays provided comparable data with a difference of <2.6%. The assays were proved to be specific, accurate and reliable, and have been used to support multiple clinical studies. The pros and cons of on-line and off-line assays with regard to man power involved in sample preparation, total analysis time, carryover, cost efficiency, and the requirement for assay transfer are discussed.     

Preparation of highly efficient monolithic silica capillary columns for the separations in weak cation-exchange and HILIC modes

Weak cation-exchange (WCX) and HILIC modes columns were prepared by on-column polymerization of acrylic acid on monolithic silica capillary columns modified with N-(3-triethoxysilylpropyl)methacrylamide anchor groups. The polymer-coated columns could be used for HILIC mode separation of pyridylamino (PA)-sugars and peptides including a tryptic digest of BSA, while for weak cation-exchange mode for the separation of proteins and nucleosides even at high linear velocity. The poly(acrylic acid) coated monolithic silica capillary columns showed greater retention toward PA-sugars than a polyacrylamide coated monolithic silica capillary columns prepared in the same manner. Proteins and nucleosides were separated effectively at pH 6.9 using the same column. The column provided fair permeability after the polymer-coating step. High-speed separation of proteins at u = 4.66 mm/s with high efficiency was shown to be possible, while high-speed separation of nucleosides has achieved within one minute using the column at u = 8.67 mm/s, suggesting that the column will be suitable for the second dimension separation of multidimensional HPLC systems.     

One-step purification of YLLIP2 isoforms from Candida sp. 99–125 by polyethyleneimine modified poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) monolith

The extracellular lipase Yarrowia lipolytica (YLLIP2) crude extract was efficiently separated and purified from Candida sp. 99–125 by one-step ion-exchange chromatography on polyethyleneimine (PEI) functionalized monolithic columns. The preparative conditions for the functionalization of monoliths were optimized, including PEI molecular mass, PEI concentration, modification time and temperature. The monolithic skeleton was prepared in situ by polymerization of glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EGDMA) with a volume ratio of 8:2. Heptane was used as the porogen. PEI 30 kDa with the concentration of 10% (v/v) was applied for the modification of the monolith at 55 °C for 12 h. Lipase (EC.3.1.1.3) from Candida sp. 99–125 was separated to four isoforms (isoform A, isoform B, isoform C and isoform D). As analyzed on non-denaturing PAGE and MALDI-TOF–MS, the four isoforms are homogenous and have the same molecular mass of approximate 38 kDa. The monoliths can afford direct crude lipase loading without increasing too much back pressure, which explores the great potential of the application of monoliths for one-single step fast separation and purification of complicated proteins.     

Optimized endodextranase-epoxy CIM® disk reactor for the continuous production of molecular weight-controlled prebiotic isomalto-oligosaccharides

An epoxy-activated monolithic Convective Interaction Media (CIM^®) disk was used for the immobilization of endodextranase D8144 from Penicillium sp. (EC 3.2.1.11) in order to produce on-line isomalto-oligosaccharides (IMOs) from Dextran T40. Enzymatic parameters, molecular weight of IMOs and performance of the IMmobilized Enzymes Reactor (IMER) were investigated. The immobilization yield of enzymes was about 45.3% (w/w), and the real specific activity close to 3.26 U mg⁻¹. The K_m values did not significantly change between free (12.8 g L⁻¹) and immobilized enzymes (14.2 g L⁻¹), due to the absence of diffusional limitation. The IMER system presented more than 80% of its residual activity after 5000 column volumes, highlighting the high stability of the immobilized endodextranases. Response surface methodology was used to enhance the performance of the IMER. Depending on dextran concentrations and flow rates, specific patterns of IMOs distributions were observed during the enzymatic hydrolysis. Finally, prebiotic activity was also investigated on IMOs produced by medium conditions (flow rate 0.3 mL min⁻¹ and dextran concentrations 4% w/w) against Lactobacillus rhamnosus GG (ATCC 53103). Their scores were at least as good as two commercialized fructo-oligosaccharides (FOS), Fibrulose^® F97 and Orafti^® P95.     

Turbulent-flow chromatography coupled on-line to fast high-performance liquid chromatography and mass spectrometry for simultaneous determination of verticine,verticinone and isoverticine in rat plasma

A method based on the on-line turbulent-flow chromatography and fast high-performance liquid chromatography/mass spectrometry (TFC–LC/MS) was developed for sensitive and high throughput pharmacokinetic study of traditional Chinese medicines (TCMs). In this method, an on-line extraction column (Waters Oasis HLB) and a fast HPLC column with sub-2 μm particle size (Agilent Zorbax StableBond-C₁₈, 4.6 mm × 50 mm, 1.8 μm) in a column-switching set-up were utilized. HLB is a reversed-phase extraction column with hydrophilic–lipophilic balanced copolymer (2.1 mm × 20 mm, 25 μm particle size), which will exhibit some turbulent-flow properties at a high-flow rate. The method combines the speed and robustness of turbulent-flow extraction and the sensitivity and separation efficiency of fast HPLC–MS to analyze multiple and trace constituents of TCMs in plasma matrix. This method was successfully applied for pharmacokinetic study of verticine, verticinone and isoverticine, the chemical markers of Fritillaria thunbergii, after oral administration of total steroidal alkaloids extract of F. thunbergii to rats. Each plasma sample was analyzed within 7 min. The method demonstrated good linearity (R > 0.999) ranged from 0.505 to 96.0 ng/mL with satisfactory accuracy and precision, and the lower limit of quantifications of verticine, verticinone and isoverticine were estimated to be 0.120, 0.595 and 0.505 ng/mL, respectively. These results indicate that the proposed method is fast, sensitive, and feasible for pharmacokinetic study of TCMs.     

Capillary liquid chromatographic analysis of fat-soluble vitamins and β-carotene in combination with in-tube solid-phase microextraction

A capillary liquid chromatography (CLC) system with UV/vis detection was coupled with an in-tube solid-phase microextraction (SPME) device for the analysis of fat-soluble vitamins and β-carotene. A monolithic silica-ODS column was used as the extraction medium. An optical-fiber flow cell with a long light path in the UV/vis detector was utilized to further enhance the detection sensitivity. In the in-tube SPME/CLC system, the pre-condition of the extraction column and the effect of the injection volume were investigated. The detection limits (LOD) for the fat-soluble vitamins and β-carotene were in the range from 1.9 to 173 ng/mL based on the signal-to-noise ratio of 3 (S/N = 3). The relative standard deviations of migration time and peak area for each analyte were less than 5.0%. The method was applied to the analysis of fat-soluble vitamins and β-carotene contents in corns.     

Resistant starch does not affect zinc homeostasis in rural Malawian children

ObjectiveThis study tested the hypothesis that Malawian children at risk for zinc deficiency will have reduced endogenous fecal zinc (EFZ) and increased net absorbed zinc (NAZ) following the addition of high amylose maize resistant starch (RS) to their diet.MethodsThis was a small controlled clinical trial to determine the effects of added dietary RS on zinc homeostasis among 17 stunted children, aged 3–5 years consuming a plant-based diet and at risk for perturbed zinc homeostasis. Dual zinc stable isotope studies were performed before and after 28 d of intervention with RS, so that each child served as their own control. The RS was incorporated into fried wheat flour dough and given under direct observation twice daily for 28 d. Changes in zinc homeostatic measures were compared using paired Student's t-tests and linear regression analysis.ResultsChildren had a mean height-for-age Z-score of −3.3, and consumed animal source foods ≤twice per month. Their habitual diet contained a phytate:zinc molar ratio of 34:1. Children avidly consumed the RS without complaints. EFZ was 0.8 ± 0.4 mg/d (mean ± SD) both before and after the intervention. Fractional absorption of zinc was 0.38 ± 0.08 and 0.35 ± 0.06 before and after the RS intervention respectively. NAZ was 1.1 ± 0.5 and 0.6 ± 0.7 before and after the RS intervention. This reduction of NAZ corresponded with diminished dietary zinc intake on the study day following intervention with RS. Regression analysis indicated no change in zinc absorption relative to dietary intake as a result of the RS intervention.ConclusionConsumption of RS did not improve zinc homeostasis in rural African children without zinc deficiency. RS was well tolerated in this setting.     

Immunoaffinity column cleanup procedure for analysis of ivermectin in swine liver

A simple and sensitive method for the analysis of ivermectin (22,23-dihydroavermectin B₁) in swine liver based on immunoaffinity column cleanup is described. The immunosorbent was prepared by coupling polyclonal anti-ivermectin antibodies to carbonyl diimidazole-activated Sepharose CL-4B. After extraction with methanol, ivermectin was cleaned up on an immunoaffinity column, and determined by reversed-phase liquid chromatography with UV absorbance detection at 245 nm. Recoveries of ivermectin from fortified samples of 5–100 μg kg⁻¹ levels ranged 85–102%, with coefficients of variation of 6–12%. The limit of detection was 2 μg kg⁻¹ in a 5-g sample.     

Impact of annealing and heat-moisture treatment on rapidly digestible,slowly digestible and resistant starch levels in native and gelatinized corn,pea and lentil starches

Impact of annealing (ANN) and heat-moisture treatment (HMT) on rapidly digestible starch (RDS), slowly digestible starch (SDS), resistant starch (RS), and expected glycemic index (eGI) of corn, pea, and lentil starches in their native and gelatinized states were determined. ANN was done for 24 h at 70% moisture at temperatures 10 and 15 °C below the onset (T_o) temperature of gelatinization, while HMT was done at 30% moisture at 100 and 120 °C for 2 h. The swelling factor (SF), amylose leaching (AML) and gelatinization parameters of the above starches before and after ANN and HMT were determined. SF and AML decreased on ANN and HMT (HMT > ANN). The gelatinization temperatures increased on ANN and HMT (HMT > ANN). However, the gelatinization temperature range decreased on ANN but increased on HMT. Birefringence remained unchanged on ANN but decreased on HMT. The Fourier transform infrared (FT-IR) absorbance ratio of 1047 cm^?1/1022 cm^?1 increased on ANN but decreased on HMT. ANN and HMT increased RDS, RS and eGI levels and decreased SDS levels in granular starches. HMT had a greater impact than ANN on RDS, RS, and SDS levels. In gelatinized starches, ANN and HMT decreased RDS and eGI, but increased SDS and RS levels. These changes were more pronounced on HMT. This study showed that amylopectin structure and interactions formed during ANN and HMT had a significant impact on RDS, SDS, RS and eGI levels of starches.     

Impact of anaerobic digestion on organic matter quality in pig slurry

Changes in pig slurry organic matter (OM) during anaerobic digestion (AD) were studied in a reactor to characterize OM evolution through AD. OM maturity and stability were evaluated using different biological and physico-chemical methods. Germination and growth chamber experiments revealed a higher maturity of digested slurry (DS) than raw slurry (RS). Soil incubations showed that DS was more stable than RS with a C-mineralization of 12.0 g CO₂-C 100 g^?1 C_org after 49 days as compared to 17.6 g CO₂-C 100 g^?1 C_org. Biochemical fractionation showed a relative increase in stable compounds such as hemicellulose-like and lignin-like molecules. Fourier-transform infrared spectroscopy showed some changes in the chemical structures of OM with a reduction in the aliphatic chain, lipid and polysaccharide levels. A comparison between the evolution of OM during AD and the first weeks of a composting process showed almost identical changes. Finally a theoretical method called Fictitious Atomic-group Separation was applied to the elemental compositions of RS and DS. DS was less humified than RS and presented the properties of a fulvic acid, indicating that the observed stability in DS was mainly due to the biodegradation of the most labile compounds.     

Automated metal-free multiple-column nanoLC for improved phosphopeptide analysis sensitivity and throughput

We report on the development and characterization of automated metal-free multiple-column nanoLC instrumentation for sensitive and high-throughput analysis of phosphopeptides with mass spectrometry. The system employs a multiple-column capillary LC fluidic design developed for high-throughput analysis of peptides (Anal. Chem. 2001, 73, 3011–3021), incorporating modifications to achieve broad and sensitive analysis of phosphopeptides. The integrated nanoLC columns (50 μm i.d. × 30 cm containing 5 μm C18 particles) and the on-line solid phase extraction columns (150 μm i.d. × 4 cm containing 5 μm C18 particles) were connected to automatic switching valves with non-metal chromatographic accessories, and other modifications to avoid the exposure of the analyte to any metal surfaces during handling, separation, and electrospray ionization. The nanoLC developed provided a separation peak capacity of ～250 for phosphopeptides (and ～400 for normal peptides). A detection limit of 0.4 fmol was obtained when a linear ion trap tandem mass spectrometer (Finnegan LTQ) was coupled to a 50-μm i.d. column of the nanoLC. The separation power and sensitivity provided by the nanoLC–LTQ enabled identification of ～4600 phosphopeptide candidates from ～60 μg COS-7 cell tryptic digest followed by IMAC enrichment and ～520 tyrosine phosphopeptides from ～2 mg of human T cells digests followed by phosphotyrosine peptide immunoprecipitation.     

Simple and selective method for the determination of various tyrosine kinase inhibitors used in the clinical setting by liquid chromatography tandem mass spectrometry

A fast, sensitive, universal and accurate method for the determination of four different tyrosine kinase inhibitors from biological material was developed using LC–MS/MS techniques. Utilizing a simple protein precipitation with acetonitrile a 20 μl sample volume of biological matrixes can be extracted at 4 °C with minimal effort. After centrifugation the sample extract is introduced directly onto the LC–MS/MS system without further clean-up and assayed across a linear range of 1–4000 ng/ml. Chromatography was performed using a Dionex Ultimate 3000 with a Phenomenex prodigy ODS3 (2.0 mm × 100 mm, 3 μm) column and eluted at 200 μl/min with a tertiary mobile phase consisting of 20 mM ammonium acetate:acetonitrile:methanol (2.5:6.7:8.3%). Injection volume varied from 0.1 μl to 1 μl depending on the concentration of the drug observed. Samples were observed to be stable for a maximum of 48 h after extraction when kept at 4 °C. Detection was performed using a turbo-spray ionization source and mass spectrometric positive multi-reaction-monitoring-mode (+MRM) for Gefitinib (447.1 m/z; 127.9 m/z), Erlotinib (393.9 m/z; 278.2 m/z), Sunitinib (399.1 m/z; 283.1 m/z) and Sorafenib (465.0 m/z; 251.9 m/z) at an ion voltage of +3500 V. The accuracy, precision and limit-of-quantification (LOQ) from cell culture medium were as follows: Gefitinib: 100.2 ± 3.8%, 11.2 nM; Erlotinib: 101.6 ± 3.7%, 12.7 nM; Sunitinib: 100.8 ± 4.3%, 12.6 nM; Sorafenib: 93.9 ± 3.0%, 10.8 nM, respectively. This was reproducible for plasma, whole blood, and serum. The method was observed to be linear between the LOQ and 4000 ng/ml for each analyte. Effectiveness of the method is illustrated with the analysis of samples from a cellular accumulation investigation and from determination of steady state concentrations in clinically treated patients.     

Chromatographic separation of (E)- and (Z)-isomers of entacapone and their simultaneous quantitation in human plasma by LC–ESI-MS/MS

A selective, sensitive and high throughput liquid chromatography–tandem mass spectrometry (LC–ESI-MS/MS) method has been developed and validated for the chromatographic separation and quantitation of (E)-entacapone and (Z)-entacapone in human plasma. Sample clean-up involved liquid–liquid extraction (LLE) of both the isomers and carbamazepine used as internal standard from 500 μL of human plasma. Both the analytes were chromatographically separated with a resolution factor of 3.0 on a Gemini C18 (50 mm × 4.6 mm, 5 μm particle size) analytical column using 1% formic acid and methanol (50:50, v/v) as the mobile phase. The selectivity factor (α) of the column for the separation was 2.0, based on the capacity factors of 2.6 and 1.3 for (E)- and (Z)-isomers respectively. The parent → product ion transitions for both the isomers (m/z 306.1 → 233.0) and IS (m/z 237.3 → 194.2) were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring (MRM) and positive ion mode. The method was validated over the concentration range of 24.3–6076 ng/mL and 23.8–5960 ng/mL for (E)-entacapone and (Z)-entacapone respectively. Matrix effect was assessed by post-column analyte infusion experiment and the process/extraction efficiency found was 94.3% and 89.3% for (E)- and (Z)-isomers respectively. The method was successfully applied to a pivotal bioequivalence study in 36 healthy human subjects after oral administration of 200 mg (E)-entacapone tablet formulation under fasting conditions.     

New monolith technology for automated anion-exchange purification of nucleic acids

Synthetic nucleic acid analysis often employs pellicular anion-exchange (AE) chromatography because it supports very high efficiency separations while offering means to control secondary structure, retention and resolution by readily modifiable chromatographic conditions. However, these pellicular anion-exchange (pAE) phases do not offer capacity sufficient for lab-scale oligonucleotide (ON) purification. In contrast, monolithic phases produce fast separations at capacities exceeding their pellicular counterparts, but do not exhibit capacities typical of fully porous, bead-based, anion-exchangers. In order to further increase monolith capacity and obtain the selectivity and mass transfer characteristics of pellicular phases, a surface-functionalized monolith was coated with pAE nanobeads (latexes) usually employed on the pellicular DNAPac phase. The nanobead-coated monolith exhibited chromatographic behaviors typical of polymer AE phases. Based on this observation the monolithic substrate surface porosity and latex diameters were co-optimized to produce a hybrid monolith harboring capacity similar to that of fully porous bead-based phases and peak shape approaching that of the pAE phases. We tested the hybrid monolith on a variety of previously developed pAE capabilities including control of ON selectivity, resolution of derivatized ONs, the ability to resolve RNA ONs harboring aberrant linkages at different positions in a single sequence and separation of phosphorothioate diastereoisomers. We compared the yield and purity of an 8 mg ON sample purified on both the new hybrid monolith and a benchmark AE column based on fully porous monodisperse beads. This comparison included an assessment of the relative selectivities of both columns. Finally, we demonstrated the ability to couple AE ON separations with ESI-MS using an automated desalting protocol. This protocol is also useful for preparing ONs for other assays, such as enzyme treatments, that may be sensitive to high salt levels.     

Monitoring of acaricide resistance of Tetranychus urticae (Acari: Tetranychidae) from Korean apple orchards

Tetranychus urticae populations were collected from ten commercial apple orchards and their susceptibilities to 12 acaricides were tested using a leaf disc bioassay. The resistance of each T. urticae population was reported as the LC₅₀ value, the resistance ratio (RR) and the slope of the probit–concentration regression. Cross resistances of T. urticae populations were estimated using the Spearman's rank correlation coefficient. Most local populations showed low resistance levels (RR ≤ 10). Development of resistance to METI and pyrethroid acaricides differed among local populations. The highest RR value (154.6) was found in the Uiseong population to tebufenpyrad. The Geochang population was highly resistant, especially to METI and pyrethroid acaricides. T. urticae populations collected from Suwon, Chungju, Yeongju and Geochang showed moderate resistance (10 < RR ≤ 40) to more than two acaricides. Resistance ratios to abamectin, chlorfenapyr, fenbutatin-oxide and milbemectin were low (RR ≤ 10) in all populations. The LC₅₀ values of abamectin, chlorfenapyr, fenbutatin-oxide and milbemectin ranged from 0.06 to 0.2 mg/l, from 0.67 to 3.38 mg/l, from 10.12 to 40.85 mg/l and from 0.47 to 3.01 mg/l, respectively. We discuss possible cross-resistance to acaricides using Spearman's rank correlation coefficient.     

A novel rapid method for simultaneous determination of eight active compounds in silymarin using a reversed-phase UPLC-UV detector

A novel rapid chromatographic method based on utilization of UPLC column was developed for the analysis of eight active compounds in silymarin. The analysis was performed on a Waters Acquity UPLC system with an Acquity UPLC^BEH C18 column (5 mm × 2.1 mm I.D., 1.7 μm) and a gradient elution of methanol and water containing 0.01% formic acid with a run time of 9 min, in which the retention time of the last analyte was 5.8 min. And all eight active compounds achieved complete separation. Comparison of system performance with conventional HPLC was made with respect to analysis time, efficiency and sensitivity. The results indicated that the type of column, the type of mobile phase and the modified addition were significant to the separation of isomeric compounds in herb extracts.     

Is T-wave alternans T-wave amplitude dependent?

The possible dependence of T-wave alternans (TWA) on T-wave amplitude was investigated in 3 orthogonal leads (X, Y, Z) 20-min resting ECG recordings and in the derived vector magnitude (VM) from 176 healthy (H) subjects and 200 coronary-artery-disease (CAD) patients. After application of our adaptive-match-filter based method for parameterization of TWA in terms of its amplitude (TWA_A) and product-magnitude (TWA_PM, defined as the product of TWA_A times TWA duration), and once a TW_A parameter was defined for T-wave amplitude quantification, the existence of intra- and inter-subjects relationships of TWA_A and TWA_PM vs. TW_A was tested. Compared to the H-population, the CAD-population showed a significant (P < 0.05) increase of TWA_A (62 ± 38 μV vs. 54 ± 25 μV) and TWA_PM (4029 ± 2974 beat μV vs. 3107 ± 1976 beat μV) and a significant decrease of TW_A (298 ± 194 μV vs. 467 ± 246 μV). These repolarization changes, however, occurred with no significant intra- or inter-subjects relationships of TWA_A and TWA_PM vs. TW_A. Thus, in our CAD and H populations there was no evidence of TWA dependence on T-wave amplitude.     

HPLC separation of some purine and pyrimidine derivatives on Chromolith Performance Si monolithic column

The chromatographic behavior of some purines and pyrimidines on a monolithic Chromolith Performance Si column under normal-phase high-performance liquid chromatography mode has been studied. Column pressure, column efficiency and selectivity of Chromolith Performance Si column were compared to those of conventional spherical 5 μm silica packed columns Econosphere Silica and Zorbax Rx-SIL. The investigation has shown that application of Chromolith Performance Si column for analysis of polar solutes can reduce the separation time without sacrificing column efficiency and selectivity. Improvement of the monolithic silica column efficiency for polar solutes is observed when ternary mobile phases (mixtures of hexane–isopropanol with ethylene glycol, water or acetonitrile) are applied.     

Simultaneous measurement of aldosterone and cortisol by high-performance liquid chromatography–tandem mass spectrometry: Application to dehydration–rehydration studies

Aldosterone and cortisol are useful biomarkers of dehydration and stress, respectively. The aim of this study was to develop an HPLC–tandem mass spectrometric method for the simultaneous measurement of aldosterone and cortisol in human plasma that could be applied to the study of athletes undergoing exercise and rehydration. Samples were prepared and analysed using an on-line sample preparation/HPLC system coupled to a triple quadrupole tandem-mass spectrometer. Samples (200 μL) were pre-treated and extracted on Hysphere C18 HD cartridges (7 μm, Spark Holland). Chromatography was performed on a Sunfire C18 analytical column (50 mm × 3.0 mm, 3 μm, Waters) under isocratic conditions at a flow rate of 0.3 mL/min. The mobile phase consisted of 35% acetonitrile/water. Mass spectrometric detection was by selected reaction monitoring using negative electrospray ionization conditions. The assay had an analytical range of 25–500 pg/mL and 25–500 ng/mL for aldosterone and cortisol, respectively (r² > 0.992, n = 22). Inter-day accuracy and imprecision for quality control samples was 99.4–106% and <16%, respectively (n = 10). In a study of nine human subjects, both aldosterone and cortisol concentrations reflected the expected physiological responses to dehydration, rehydration and exercise when measured by this method. The reported method is suitable to facilitate the study of athletes undergoing dehydration and rehydration protocols.