Validation and application of a 96-well format solid-phase extraction and liquid chromatography-tandem mass spectrometry method for the quantitation of digoxin in human plasma

To evaluate the pharmacokinetics of digoxin in humans, a sensitive and specific LC/MS/MS method was developed and validated for the determination of digoxin concentrations in human plasma. The method was shown to be more sensitive, specific, accurate, and reproducible than common techniques such as RIA. For detection, a LC/MS/MS system with electro spray ionization tandem mass spectrometry in the positive ion-multiple reaction-monitoring (MRM) mode was used to monitor precursor to product ions of m/z 798.5-51.5 for digoxin and m/z 782.5-35.5 for the internal standard, digitoxin. The method was validated over a concentration range of 0.02-5 ng/mL and was found to have acceptable accuracy, precision, linearity, and selectivity. The mean extraction recovery from spiked plasma samples was above 80%. Imidafenacin, coadministered in a drug-drug interaction study, had no detectable influence on the determination of digoxin in human plasma. The novel method was applied to a drug-drug interaction study of digoxin and imidafenacin and the characterization of steady-state pharmacokinetics of digoxin in humans after oral administration at a dose of 0.25 mg on days 1 and 2 followed by 0.125 mg daily doses on days 3 through 8.     

Specific method for determination of gefitinib in human plasma, mouse plasma and tissues using high performance liquid chromatography coupled to tandem mass spectrometry

A rapid, sensitive and specific method was developed and validated using liquid chromatography-tandem mass spectrometry (LC/MS/MS) for determination of gefitinib in human plasma and mouse plasma and tissue. Sample preparation involved a single protein precipitation step by the addition of 0.1 mL of plasma or a 200 mg/mL tissue homogenate diluted 1/10 in human plasma with 0.3 mL acetonitrile. Separation of the compounds of interest, including the internal standard (d8)-gefitinib, was achieved on a Waters X-Terra C18 (50 mm x 2.1 mm i.d., 3.5 microm) analytical column using a mobile phase consisting of acetonitrile-water (70:30, v/v) containing 0.1% formic acid and isocratic flow at 0.15 mL/min for 3 min. The analytes were monitored by tandem mass spectrometry with electrospray positive ionization. Linear calibration curves were generated over the range of 1-1000 ng/mL for the human plasma samples and 5-1000 ng/mL for mouse plasma and tissue samples with values for the coefficient of determination of > 0.99. The values for both within- and between-day precision and accuracy were well within the generally accepted criteria for analytical methods (< 15%). This method was subsequently used to measure concentrations of gefitinib in mice following administration of a single dose of 150 mg/kg intraperitoneally and in cancer patients receiving an oral daily dose of 250 mg.     

Development and validation of a sensitive LC/MS/MS method for the simultaneous determination of naloxone and its metabolites in mouse plasma

A rapid, specific, and reliable LC-MS/MS based bioanalytical method was developed and validated for the simultaneous determination of naloxone (NLX) and its two metabolites, 6β-naloxol (NLL) and naloxone-3β-D-glucuronide (NLG) in mouse plasma. The optimal chromatographic behavior of these analytes was achieved on an Aquasil C18 column (50 mm × 2.1 mm, 5 μm) using reversed phase chromatography. The total LC analysis time per injection was 2.5 min with a flow rate of 1.0 mL/min with gradient elution. Sample preparation via protein precipitation with acetonitrile in a 96-well format was applied for analyses of these analytes. The analytes were monitored by electrospray ionization in positive ion multiple reaction monitoring (MRM) mode. Modification of collision energy besides chromatographic separation was applied to further eliminate interference peaks for NLL and NLG. The method validation was conducted over the curve range of 0.200/0.400/0.500 to 100/200/250 ng/mL for NLX/NLL/NLG, respectively, using 0.0250 mL of plasma sample. The intra- and inter-day precision and accuracy of the quality control samples at low, medium, and high concentration levels showed ≤ 6.5% relative standard deviation (RSD) and -8.3 to -2.5% relative error (RE). The method was successfully applied to determine the concentrations of NLX, NLL, and NLG in incurred mouse plasma samples.     

Rapid and simultaneous determination of tacrolimus (FK506) and diltiazem in human whole blood by liquid chromatography-tandem mass spectrometry: application to a clinical drug-drug interaction study

Tacrolimus (FK506) is a potent immunosuppressant widely used for organ transplantation patients while diltiazem (DTZ), a calcium-channel inhibitor, is often used in renal transplantation patients to prevent post-transplant hypertension. However, DTZ has a significant pharmacokinetic interaction with FK506. In this study, a rapid and sensitive ammonium-adduct based liquid chromatography-tandem mass spectrometry (LC/MS/MS) method has been developed and validated for the simultaneous determination of FK506 and DTZ in human whole blood using ascomycin as the internal standard (IS). After extraction of the whole blood samples by ethyl acetate, FK506, DTZ and the IS were subjected to LC/MS/MS analysis using electro-spray positive-ion mode ionization (ESI(+)). Chromatographic separation was performed on a Hypersil BDS C18 column (50 mm x 2.1 mm, i.d., 3 microm). The MS/MS detection was conducted by monitoring the fragmentation of 821.7-->768.9 (m/z) for FK506, 415.5-->310.3 (m/z) for DTZ and 809.8-->757.0 (m/z) for IS. The method had a chromatographic running time of approximately 2 min and linear calibration curves over the concentrations of 0.5-200 ng/mL for FK506 and 2-250 ng/mL for DTZ. The recoveries of liquid-liquid extraction method were 58.3-62.6% for FK506 and 50.4-58.8% for DTZ. The lower limit of quantification (LLOQ) of the analytical method was 0.5 ng/mL for FK506 and 2 ng/mL for DTZ. The intra- and inter-day precision was less than 15% for all quality control samples at concentrations of 2, 10, and 50 ng/mL for FK506 and 5, 25, and 100 ng/mL for DTZ. The validated LC/MS/MS method has been successfully used to analyze the concentrations of FK506 and DTZ in whole blood samples from pharmacokinetic studies in renal transplanted patients.     

Rapid and simultaneous determination of nifedipine and dehydronifedipine in human plasma by liquid chromatography-tandem mass spectrometry: Application to a clinical herb-drug interaction study

Nifedipine (NIF), a calcium channel antagonist, is metabolized primarily by cytochrome P450 (CYP3A4) to dehydronifedipine (DNIF). As such, NIF is often used as a probe drug for determining CYP3A4 activity in human studies. A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed and validated to simultaneously determine NIF and DNIF in human plasma using nitrendipine as the internal standard (IS). After extraction of the plasma samples by ether-n-hexane (3:1, v/v), NIF, DNIF and the IS were subjected to LC/MS/MS analysis using electro-spray ionization (ESI). Chromatographic separation was performed on a Hypersil BDS C(18) column (50 mm x 2.1 mm, i.d., 3 microm). The method had a chromatographic running time of approximately 2.5 min and linear calibration curves over the concentrations of 0.5-100 ng/mL for NIF and DNIF. The recoveries of the one-step liquid extraction method were 81.3-89.1% for NIF and 71.6-80.4% for DNIF. The lower limit of quantification (LLOQ) of the analytical method was 0.5 ng/mL for both analytes. The intra- and inter-day precision was less than 15% for all quality control samples at concentrations of 2, 10, and 50 ng/mL. The validated LC/MS/MS method has been successfully used to study pharmacokinetic interactions of NIF with the herbal antidepressant St. John's wort in healthy volunteers. These results indicated that the developed LC/MS/MS method was efficient with a significantly shorter running time (2.5 min) for NIF and DNIF compared to those methods previously reported in the literature. The presented LC/MS/MS method had acceptable accuracy, precision and sensitivity and was used in a clinical pharmacokinetic interaction study of NIF with St. John's wort, a known herbal inducer of CYP3A4. St. John's wort was shown to induce NIF metabolism with increased plasma concentrations of DNIF.     

Development and validation of a selective and robust LC-MS/MS method for high-throughput quantifying rizatriptan in small plasma samples: application to a clinical pharmacokinetic study

An analytical method based on liquid chromatography with positive ion electrospray ionization (ESI) coupled to tandem mass spectrometry detection (LC-MS/MS) was developed for the determination of a potent 5-HT(1B/1D) receptor agonist, rizatriptan in human plasma using granisetron as the internal standard. The analyte and internal standard were isolated from 100 microL plasma samples by liquid-liquid extraction (LLE) and chromatographed on a Lichrospher C18 column (4.6mm x 50mm, 5 microm) with a mobile phase consisting of acetonitrile-10mM aqueous ammonium acetate-acetic acid (50:50:0.5, v/v/v) pumped at 1.0 mL/min. The method had a chromatographic total run time of 2 min. A Varian 1200 L electrospray tandem mass spectrometer equipped with an electrospray ionization source was operated in selected reaction monitoring (SRM) mode with the precursor-to-product ion transitions m/z 270-->201 (rizatriptan) and 313.4-->138 (granisetron) used for quantitation. The assay was validated over the concentration range of 0.05-50 ng/mL and was found to have acceptable accuracy, precision, linearity, and selectivity. The mean extraction recovery from spiked plasma samples was above 98%. The intra-day accuracy of the assay was within 12% of nominal and intra-day precision was better than 13% C.V. Following a 10mg dose of the compound administered to human subjects, mean concentrations of rizatriptan ranged from 0.2 to 70.6 ng/mL in plasma samples collected up to 24h after dosing. Inter-day accuracy and precision results for quality control samples run over a 5-day period alongside clinical samples showed mean accuracies of within 12% of nominal and precision better than 9.5% C.V.     

Development and validation of a rapid 96-well format based liquid-liquid extraction and liquid chromatography-tandem mass spectrometry analysis method for ondansetron in human plasma

A rapid and sensitive LC-MS/MS method for the quantification of ondansetron was developed and validated. The plasma samples were treated by a semi-automated liquid-liquid extraction (LLE) in 1.2 mL 96-well format micro-tubes. Ondansetron and the internal standard (IS) granisetron were analyzed by combined reversed phase LC-MS/MS, with positive ion electrospray ionization, using multiple reactions monitoring (MRM). The statistical evaluation for this method reveals excellent linearity, accuracy and precision values for the range of concentrations 0.25-40.0 ng/mL. The proposed method enabled the reliable determination of ondansetron in bioequivalence studies after per os administration of a 4 or 8 mg tablet.     

Comparative studies on the biophysical activities of the low-molecular-weight hydrophobic proteins purified from bovine pulmonary surfactant

Two low-molecular-weight hydrophobic proteins with nominal molecular weights Mr = 15,000 and Mr = 3,500 have been isolated from the lipid extracts of bovine pulmonary surfactant by several methods, including (a) dialysis plus silicic acid chromatography, (b) elution from Waters SEP-PAK silica cartridges with a variety of solvent mixtures, and (c) ultrafiltration. As detailed in the text, these proteins have been designated surfactant-associated protein-BC (SP-BC) (15 kDa: nonreduced), and SP-C (3.5 kDa). The biophysical activities of reconstituted surfactant containing these proteins and the phospholipids present in lung surfactant have been compared with the biophysical activities of bovine lipid extract surfactant on a pulsating bubble surfactometer using a phospholipid concentration of 10 mg/ml. At this concentration, unmodified lipid extract surfactant reduces the surface tension of the pulsating bubble to near 0 within 10 pulsations at 20 cycles per min. Similar biophysical properties were observed with modified lipid extract surfactant in which the relative concentration of hydrophobic protein had been reduced from 1 to 0.4% (W/W) of the phospholipids by addition of dipalmitoylphosphatidylcholine (DPPC) or DPPC plus phosphatidylglycerol. Reconstituted surfactants, which contained partially delipidated SP-BC (15 kDa: nonreduced) obtained by method (a) at a relative concentration of 0.1%, were also capable of reducing the surface tension to near 0 mN/m. Preparations of SP-BC (15 kDa: nonreduced) obtained by method (b), which had been subjected to very low pH levels during isolation and were extensively delipidated, exhibited full biophysical activity only at higher protein concentrations and with prolonged pulsation. Extensively delipidated samples of SP-BC obtained by method (c) exhibited impaired biophysical activities, even when prepared with neutral organic solvents. Reconstituted surfactant samples containing SP-C (3.5 kDa) obtained by any of the methods listed above were only able to reduce the surface tension at minimum bubble radius to approx. 20 mN/m. The biophysical activity of SP-C (3.5 kDa) was not significantly affected by low pH or extensive delipidation. Reconstituted samples containing mixtures of SP-BC (15 kDa: nonreduced) and SP-C (3.5 kDa) were more effective than samples containing either protein alone. Furthermore, with samples containing both hydrophobic proteins the final surface tensions at maximum bubble radius were attained within a few bubble pulsations.(ABSTRACT TRUNCATED AT 400 WORDS)     

Colloidal gas aphrons: A novel approach to protein recovery

Sebba (1987) defined colloidal gas aphrons (CGA) as microbubbles stabilized by surfactant layers, which are created by stirring surfactant solutions at speeds greater than a critical value. A high shear impeller is used for stirring and critical values for the impeller speed must be exceeded to create these stable gas liquid dispersions (typically >5000 rpm). Although there have been no previous reports of direct protein recovery using CGA, it is likely that, with appropriate choice of surfactant, proteins should adsorb to these surfactant bubbles by means of electrostatic and/or hydrophobic interactions. This is the basis of this study, in which the use of CGA for protein recovery from aqueous solution is considered. A surfactant which has been characterized previously for generation of CGA was chosen (Jauregi et al., 1997), i.e., the anionic surfactant sodium bis-(2-ethyl hexyl) sulfosuccinate (AOT). Lysozyme, a well-characterized protein, was chosen as the protein to be recovered. Lysozyme was recovered successfully from aqueous solution using CGA generated from AOT. At optimum conditions, lysozyme recovery, enrichment ratio, and separation ratio were 95%, 19 and 302 respectively, with enzyme activity maintained. These results indicate the exciting potential of this technique. A wide range of process conditions including initial concentration of protein and surfactant, surfactant/protein molar ratio, pH, and ionic strength were considered. High recoveries and enrichments were generally obtained at protein concentrations 0.11 mg/mL. However, at high ionic strength (0.29M) poor separation and recoveries were obtained at low protein concentrations (counter-ions diminishing electrostatic interactions between protein and aphrons at this condition). In general, (ns/np)a was determined to be between 10 and 16 for experiments in which high levels of recovery/separation parameters were found. For most conditions, protein precipitation was observed; however, this precipitate could be resolubilized without loss of enzyme activity.     

Application of a sensitive liquid chromatographic/tandem mass spectrometric method to pharmacokinetic study of nalmefene in humans

A sensitive, specific and rapid liquid chromatographic/tandem mass spectrometric (LC/MS/MS) method was developed and validated for quantification of nalmefene in human plasma. An aliquot of 200 microL plasma sample was simply precipitated by 400 microL methanol. Separation of nalmefene and the internal standard hydromorphone from the interferences was achieved on a C(18) column followed by MS/MS detection. The analytes were monitored in the positive ionization mode with a TurboIonspray source. The method had a total chromatographic run time of 4.5 min and linear calibration curves over the concentration range of 10-5000 pg/mL. The lower limit of quantification (LLOQ) was 10 pg/mL. The intra- and inter-day precision was less than 10.1% determined from QC samples at concentrations of 30, 300 and 4500 pg/mL, and the accuracy was within +/-3.4%. As the method was more sensitive (10 times higher) than those reported previously, we investigated the pharmacokinetics of nalmefene in healthy volunteers after a single intravenous injection of low dose (30 microg) of nalmefene hydrochloride for the first time.     

Use of sodium lauroyl sarcosinate in a high-sensitivity protein assay by resonance light scattering technique

A simple and high-sensitivity method has been developed for the determination of proteins in aqueous solutions by resonance light scattering (RLS) technique. At pH 3.4 and ionic strength 1.2 x 10(-3), the weak RLS intensity of sodium lauroyl sarcosinate was greatly enhanced by the addition of proteins with the maximum peak located at 391 nm. Under the optimum conditions, the enhanced RLS intensities were in proportion to the concentrations of proteins in the range of 0.04 to 2.1 microg/mL for lysozyme, 0.0025 to 1.2 microg/mL for bovine serum albumin, 0.0075 to 0.9 microg/mL for human serum albumin, 0.02 to 1.4 microg/mL for gamma-globulin, 0.02 to 0.8 microg/mL for egg albumin, and 0.01 to 0.6 microg/mL for hemoglobin. Low detection limits ranging from 0.8 ng/mL to 4.3 ng/mL depending on the kind of proteins that have been achieved. The protein concentrations in synthetic samples and real biochemical samples were determined with satisfactory results. This method presented here is not only sensitive and simple but also reliable and suitable for practical bioassay applications.     

Validated liquid chromatography–tandem mass spectrometry method for quantitative determination of dauricine in human plasma and its application to pharmacokinetic study

A highly sensitive and selective LC–MS/MS method was developed and validated for the determination of dauricine in human plasma, using protopine as internal standard (IS). The analyte and IS were extracted by liquid–liquid extraction and analyzed by LC–MS/MS. Chromatographic separation was performed on Agilent TC-C₁₈ column with a mobile phase of methanol–water–glacial acetic acid (60:40:0.8, v/v/v) at a flow rate of 0.7 mL/min. Detection was performed on a triple quadrupole tandem mass spectrum by multiple reaction monitoring (MRM) mode using the electrospray ionization technique in positive mode. The method was linear over the concentration range of 1–200 ng/mL. The lower limit of quantification (LLOQ) was 1 ng/mL in human plasma with acceptable precision and accuracy. The intra- and inter-day precision was less than 5.9% determined from quality control (QC) samples at concentrations of 2.0, 20.0 and 160 ng/mL, and the accuracy was within ±9.9%. This method was successfully applied for the evaluation of pharmacokinetics of dauricine after oral doses of 100, 300 and 600 mg phenolic alkaloids of menispermum dauricum tablet (PAMDT) to 12 Chinese healthy volunteers.     

Simultaneous determination of azelnidipine and two metabolites in human plasma using liquid chromatography-tandem mass spectrometry

A quantitative assay method by liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) for the simultaneous determination of azelnidipine and its two metabolites, M-1 (aromatized form) and M-2 (hydroxylated form), in human plasma was developed and validated. Plasma samples, each of 1.0mL, were extracted by a single step liquid-liquid extraction using a mixture of ethyl acetate and hexane (1:1, v/v), and analyzed by the LC/ESI-MS/MS method. Three analytes were separated by isocratic elution on a C(18) column, and ionized using a positive ion electrospray ionization source. The ion transitions were monitored in selected reaction monitoring (SRM) mode. The chromatographic run time was 11min per injection, with retention time of 3.6, 10.2 and 6.8min for azelnidipine, M-1 and M-2, respectively. The calibration curves for azelnidipine, M-1 and M-2 well fitted to equations by a weighted (1/X(2)) quadratic regression over the range of 0.5-40.0ng/mL (r(2)>0.9979). The intra- and inter-assay precisions (coefficient of variation: C.V.), calculated from quality control (QC) samples, were less than 8.7 and 8.4%, 3.8 and 4.7%, and 11.9 and 13.9%, respectively, for azelnidipine, M-1 and M-2. The accuracy was within +/-9% for azelnidipine, within +/-7% for M-1 and within +/-16% for M-2. The overall recoveries for azelnidipine, M-1 and M-2 were 68.8-78.6%, 54.3-62.9% and 80.4-89.7%, respectively. All analytes evaluated demonstrated acceptable short-term, long-term, auto-sampler and stock solution stabilities. Furthermore, the method developed was successfully applied to pharmacokinetic studies on azelnidipine, M-1 and M-2 after an oral dose of 16mg CALBLOCK tablets (2mgx8mg tablets) to healthy volunteers.     

LC/MS/MS measurement of gentamicin in bovine plasma, urine, milk, and biopsy samples taken from kidneys of standing animals

Methods for the measurement of gentamicin concentration in several bovine tissues were developed and validated. A novel liquid chromatographic (LC) technique employed trifluoroacetic acid in the mobile phase so that all gentamicin components co-eluted. Analytes were ionized by positive-ion pneumatically assisted electrospray and detected by selected reaction monitoring (SRM) with an LC-tandem mass spectrometer (LC/MS/MS). Calibration of plasma and urine samples was based on tobramycin internal standard. Calibration of milk and kidney samples was based on external standard, due to variability of tobramycin response in these matrices. The extraction technique employed treatment with aqueous trichloroacetic acid to both precipitate protein and liberate gentamicin from the matrix. Milk samples had to be defatted by centrifugation prior to extraction. Urine samples were further cleaned up with C-18 solid phase extraction (SPE). These methods were validated for use in several residue depletion studies (reported elsewhere) to monitor the depletion of gentamicin in tissues under various dosing conditions. The plasma method was calibrated from 1 to 5000 ng/mL in two ranges, with a limit of quantitation (LOQ) in the low range calculated at 3.3 ng/mL. The milk method was calibrated from 2.5 to 2500 ng/mL with an LOQ calculated at 4.5 ng/mL. The urine method was designed for use at low levels, and was calibrated from 1 to 100 ng/mL with an LOQ of 3.8 ng/mL. The kidney method was primarily designed for analysis of small samples (approximately 100mg). This method was calibrated from 10 to 50,000 ng/g with an LOQ of 26 ng/g.     

Investigation of high-throughput ultrafiltration for the determination of an unbound compound in human plasma using liquid chromatography and tandem mass spectrometry with electrospray ionization

A high-throughput ultrafiltration method with a direct injection assay has been developed to determine unbound concentrations of a high-protein binding compound, an alpha(v)beta(3) bone integrin antagonist (I), in human plasma for a clinical pharmacokinetic study. The 96-well MultiScreen filter plate with Ultracel-PPB membrane was evaluated for the separation of unbound from protein-bound compound I by ultrafiltration. The sample preparation was automated using a Packard MultiPROBE II EX liquid handling system to transfer the plasma samples to the 96-well PPB plate for centrifugation and to prepare ultrafiltrate samples for analysis. Using on-line extraction with a column-switching setup for sample clean-up and separation, the ultrafiltrate samples were directly injected onto a reversed-phase HPLC system and analyzed using a mass spectrometer interfaced with an electrospray ionization (ESI) source in the positive ionization mode (LC/ESI-MS/MS). The performance of the ultrafiltration using Ultracel-PPB 96-well plate for unbound I analysis was evaluated and optimized with respect to sample volume, centrifugation temperature, speed and time, and the relationship of the well positions of the PPB plate versus filtrate volumes and concentrations. The assay intraday accuracy and precision were between 93.9 and 104.8 and <7.3% (CV), respectively. The linear range of the calibration curve for the assay was 0.1-500 ng/mL on a Finnigan TSQ Quantum LC/ESI-MS/MS system. Evaluation and validation of the unbound plasma assay demonstrated it to be rapid, sensitive and reproducible.     

Liquid chromatography MALDI MS/MS for membrane proteome analysis

Membrane proteins play critical roles in many biological functions and are often the molecular targets for drug discovery. However, their analysis presents a special challenge largely due to their highly hydrophobic nature. We present a surfactant-aided shotgun proteomics approach for membrane proteome analysis. In this approach, membrane proteins were solubilized and digested in the presence of SDS followed by newly developed auto-offline liquid chromatography/matrix-assisted laser desorption ionization (LC/MALDI) tandem MS analysis. Because of high tolerance of MALDI to SDS, one-dimensional (1D) LC separation can be combined with MALDI for direct analysis of protein digests containing SDS, without the need for extensive sample cleanup. In addition, the heated droplet interface used in LC/MALDI can work with high flow LC separations, allowing a relatively large amount of protein digest to be used for 1D LC/MALDI which facilitates the detection of low abundance proteins. The proteome identification results obtained by LC/MALDI are compared to the gel electrophoresis/MS method as well as the shotgun proteomics method using 2D LC/electrospray ionization MS. It is demonstrated that, while LC/MALDI provides more extensive proteome coverage compared to the other two methods, these three methods are complementary to each other and a combination of these methods should provide a more comprehensive membrane proteome analysis.     

In-tube solid-phase microextraction coupled to liquid chromatography (in-tube SPME/LC) analysis of nontricyclic antidepressants in human plasma

A sensitive, selective, and reproducible in-tube solid-phase microextraction and liquid chromatographic (in-tube SPME/LC-UV) method for simultaneous determination of mirtazapine, citalopram, paroxetine, duloxetine, fluoxetine, and sertraline in human plasma was developed, validated and further applied to the analysis of plasma samples from elderly patients undergoing therapy with antidepressants. Important factors in the optimization of in-tube SPME efficiency are discussed, including the sample draw/eject volume, draw/eject cycle number, draw/eject flow-rate, sample pH, and influence of plasma proteins. The quantification limits of the in-tube SPME/LC method varied between 20 and 50ng/mL, with a coefficient of variation lower than 10%. The response of the in-tube SPME/LC method for most of the drugs was linear over a dynamic range from 50 to 500ng/mL, with correlation coefficients higher than 0.9985. The in-tube SPME/LC can be successfully used to analyze plasma samples from ageing patients undergoing therapy with nontricyclic antidepressants.     

Application of a sensitive liquid chromatographic/tandem mass spectrometric method to a pharmacokinetic study of allylestrenol in healthy Chinese female volunteers

A sensitive, specific and rapid liquid chromatographic/tandem mass spectrometric (LC/MS/MS) assay for the determination of allylestrenol in human plasma was established. Plasma samples were extracted by tert-butyl ether and separated by LC/MS/MS using a Phenomenex Curosil-PFP column (250 mm x 4.6 mm ID, dp 5 microm) with a mobile phase of methanol-water (95:5, v/v). The analytes were monitored with atmospheric pressure chemical ionization (APCI) by selected reaction monitoring (SRM) mode. The linear calibration curves covered a concentration range of 0.04-20.0 ng/mL with lower limit of quantification (LLOQ) at 0.04 ng/mL. The mean extraction recovery of allylestrenol was greater than 81.8%. The intra- and inter-day precisions were less than 1.3% and 3.1% respectively, determined from quality control (QC) samples of three representative concentrations. The method has been successfully applied to determining the plasma concentration of allylestrenol and a clinical pharmacokinetics study in healthy Chinese female volunteers.     

Giant extracellular Glossoscolex paulistus Hemoglobin (HbGp) upon interaction with cethyltrimethylammonium chloride (CTAC) and sodium dodecyl sulphate (SDS) surfactants: Dissociation of oligomeric structure and autoxidation

The effects of two ionic surfactants on the oligomeric structure of the giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) in the oxy - form have been studied through the use of several spectroscopic techniques such as electronic optical absorption, fluorescence emission, light scattering, and circular dichroism. The use of anionic sodium dodecyl sulphate (SDS) and cationic cethyltrimethyl ammonium chloride (CTAC) has allowed to differentiate the effects of opposite headgroup charges on the oligomeric structure dissociation and hemoglobin autoxidation. At pH 7.0, both surfactants induce the protein dissociation and a significant oxidation. Spectral changes occur at very low CTAC concentrations suggesting a significant electrostatic contribution to the protein–surfactant interaction. At low protein concentration, 0.08 mg/ml, some light scattering within a narrow CTAC concentration range occurs due to protein–surfactant precipitation. Light scattering experiments showed the dissociation of the oligomeric structure by SDS and CTAC, and the effect of precipitation induced by CTAC. At higher protein concentrations, 3.0 mg/ml, a precipitation was observed due to the intense charge neutralization upon formation of ion pair in the protein–surfactant precipitate. The spectral changes are spread over a much wider SDS concentration range, implying a smaller electrostatic contribution to the protein–surfactant interactions. The observed effects are consistent with the acid isoelectric point (pI) of this class of hemoglobins, which favors the intense interaction of HbGp with the cationic surfactant due to the existence of excess acid anionic residues at the protein surface. Protein secondary structure changes are significant for CTAC at low concentrations while they occur at significantly higher concentrations for SDS. In summary, the cationic surfactant seems to interact more strongly with the protein producing more dramatic spectral changes as compared to the anionic one. This is opposite as observed for several other hemoproteins. The surfactants at low concentrations produce the oligomeric dissociation, which facilitates the iron oxidation, an important factor modulating further oligomeric protein dissociation.     

Surfactant effects on protein structure examined by electrospray ionization mass spectrometry.

Electrospray ionization mass spectrometry (ESI-MS) has proven to be a useful tool for examining noncovalent complexes between proteins and a variety of ligands. It has also been used to distinguish between denatured and refolded forms of proteins. Surfactants are frequently employed to enhance solubilization or to modify the tertiary or quaternary structure of proteins, but are usually considered incompatible with mass spectrometry. A broad range of ionic, nonionic, and zwitterionic surfactants was examined to characterize their effects on ESI-MS and on protein structure under ESI-MS conditions. Solution conditions studied include 4% acetic acid/50% acetonitrile/46% H2O and 100% aqueous. Of the surfactants examined, the nonionic saccharides, such as n-dodecyl-beta-D-glucopyranoside, at 0.1% to 0.01% (w/v) concentrations, performed best, with limited interference from chemical background and adduct formation. Under the experimental conditions used, ESI-MS performance in the presence of surfactants was found to be unrelated to critical micelle concentration. It is demonstrated that surfactants can affect both the tertiary and quaternary structures of proteins under conditions used for ESI-MS. However, several of the surfactants caused significant shifts in the charge-state distributions, which appeared to be independent of conformational effects. These observations suggest that surfactants, used in conjunction with ESI-MS, can be useful for protein structure studies, if care is used in the interpretation of the results.