Quantitative determination of salidroside in rat plasma by on-line solid-phase extraction integrated with high-performance liquid chromatography/electrospray ionization tandem mass spectrometry

A method for the quantification of salidroside, a major biologically active compound in Rhodiola, in rat plasma by on-line SPE LC/MS/MS in negative electrospray mode was developed and validated. A column-switching instrument and two HPLC pumping systems were employed, and salicin was used as the internal standard. A Waters Oasis HLB extraction column and an Agilent TC-C(18) analytical column in a column-switching set-up with gradient elution were utilized. The MS/MS ion transitions monitored were m/z 299.0/119.0 and 285.1/122.9 for salidroside and salicin, respectively. The standard curves were linear within a range of 50-5000 ng/mL using weighted linear regression analysis (1/x). The intra- and inter-day coefficients of variance ranged from 1% to 9%. The recovery was above 90%. The freeze/thaw and long-term stability were validated. This method was subsequently applied to a pharmacokinetic study of salidroside in rats.     

Analysis of urinary aristolactams by on-line solid-phase extraction coupled with liquid chromatography-tandem mass spectrometry

Aristolochic acids (AAs), nephrotoxicants and known human carcinogens, are a mixture of structurally related derivatives of nitrophenanthrene carboxylic acids with the major components being aristolochic acid I and aristolochic acid II. People may ingest small amounts of AAs from its natural presence in medicinal plants and herbs of the family Aristolochiaceae, including the genera Aristolochia and Asarum, which have been used worldwide in folk medicine for centuries. In order to assess AA intake, an on-line solid-phase extraction coupled with liquid chromatography-tandem mass spectrometry (on-line SPE-LC/MS/MS) method was developed to analyze their most abundant corresponding metabolites, aristolactams (ALs), in urine to serve as biomarkers. The limits of quantitation were 0.006 ng for aristolactam I (AL-I), and 0.024 ng for aristolactam II (AL-II) on column. Recovery varied from 98.0% to 99.5%, and matrix effects were within 75.3-75.4%. This method was applied to analyze ALs in the urine samples collected on days 1, 2, 4, and 7 from mice treated with 30 mg/kg or 50mg/kg AAs. Their half lives were estimated to be 3.55 h and 4.00 for AL-I, and 4.04 and 4.83 h for AL-II, depending on AAs doses. These results demonstrated that the first simple on-line SPE-LC/MS/MS method was successfully developed to analyze urinary ALs with excellent sensitivity and specificity to serve as biomarkers to assess current AA intake from AAs-containing Chinese herbs.     

Quantification of puerarin in plasma by on-line solid-phase extraction column switching liquid chromatography-tandem mass spectrometry and its applications to a pharmacokinetic study

A highly precise, automatic and rapid method for quantification of puerarin in canine and human plasma using an on-line solid-phase extraction (SPE) column switching procedure combined with liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS) was developed. The eluent of SPE column consisted of acetonitrile/methanol/0.1% formic acid (25/25/50) at a flow rate of 0.2mLmin(-1). Puerarin was analyzed by a linear ion trap mass spectrometer, LTQ-MS, operating in the negative ion and selective reaction monitoring (SRM) acquisition mode. Method validation results demonstrated that the linear calibration curve covered a wide range of 0.39-400.00ngmL(-1), the correlation coefficients (r(2)) were above 0.999. The lower limit of detection (LLOD) with the signal-to-noise (S/N) ratio higher than 12 was 0.39ngmL(-1). The intra- and inter-batch precisions were less than 7.61% and 6.42%, respectively. The accuracy was well within the accept limit. The on-line SPE column switching HPLC-MS system was applied to pharmacokinetic (PK) study of puerarin after a single orally dose in beagles. And the optimum conditions were successfully utilized to quantify puerarin in human plasma, which indicated the feasibility and the reliability of this method for application in preclinical and clinical PK studies of isoflavone drugs.     

Rapid and highly automated determination of morphine and morphine glucuronides in plasma by on-line solid-phase extraction and column liquid chromatography

A high-performance liquid chromatography (HPLC) method has been developed for the determination of morphine and its main metabolites, morphine-6-glucuronide (M-6-G) and morphine-3-glucuronide (M-3-G), in plasma or cerebrospinal fluid. Samples were extracted using on-line solid-phase extraction followed by reversed-phase HPLC with fluorescence detection. Recoveries of 20 ng morphine and morphine glucuronides in plasma were over 95%. The limit of detection using 400 μl of a biological matrix was 0.85, 3.4 and 1.0 ng/ml of M-3-G, M-6-G and morphine, respectively. Inter- and intra-day assay precision was better than 10%. The main advantages of the present described method are increased recoveries (>95%) and a high degree of automation allowing a high speed in routine analysis. The time required for the fully automated analysis of one sample was less than 26 min.     

Measurement of anti-cancer agent methoxyamine in plasma by tandem mass spectrometry with on-line sample extraction

In this work, we present the development and validation of a tandem mass spectrometry method for the quantitative determination of methoxyamine (CH3ONH2), a potential new chemotherapeutic agent, in human and mouse plasma. Methoxyamine together with the internal standard (I.S.) methoxyl-D3-amine was directly derivatized in plasma sample with a novel chemical agent 4-(N,N-diethylamino)benzaldehyde. The product solution was injected into an on-line Oasis HLB extraction column (2.1 mm x 20 mm) for analyte extraction. After the elution of extractives, the derivatized analytes were monitored by the positive-electrospray-ionization mass spectrometry (ESI-MS-MS). The structures of derivatized analytes were elucidated by fragmentation. Quantitation of plasma methoxyamine was carried out by the multiple reaction monitoring (MRM) mode. This method had a linear calibration range of 1.00-1000 ng/ml with a correlation coefficient of 0.9999 for methoxyamine in both human and mouse plasma. The limit of detection (LOD) and limit of quantification (LOQ) for methoxyamine in plasma were 0.150 and 0.500 ng/ml, respectively. It was demonstrated that the method had high recovery and accuracy (90.1-94.7 and 90.1-96.3%), as well as excellent intra- and inter-assay precision (2.2 and 3.7%), at three concentration levels (5.00, 50.0, 500 ng/ml). This method has been used to analyze the plasma levels of methoxyamine in samples obtained from male CD1 mice after bolus intraperitoneal injection of 2, 5 and 20mg methoxyamine hydrochloride (CH3ONH2.HCl) per kilogram mouse.     

Enantiomeric determination of amlodipine in human plasma by liquid chromatography coupled to tandem mass spectrometry

A sensitive method for the separation and determination of amlodipine enantiomers in plasma has been developed based on solid-phase extraction (SPE) with disposable extraction cartridges (DECs) in combination with chiral liquid chromatography (LC). The SPE technique is used to isolate the drug from the biological matrix and to prepare a cleaner sample before injection and analysis by HPLC coupled to mass spectrometry. The DEC is filled with ethyl silica (50 mg) and is first conditioned with a 2.5% ammonia in methanol solution and then with ammonium acetate buffer. A 1.0-ml volume of plasma is then applied on the DEC. The washing step is first performed with ammonium acetate buffer and secondly with a mixture of water and methanol (65:35, v/v), while the final elution step is obtained by dispensing methanol containing 2.5% of ammonia. The eluate is then collected and evaporated to dryness before being dissolved in the LC mobile phase and injected into the LC system. The stereoselective analysis of amlodipine is achieved on a Chiral AGP column containing alpha(1)-acid glycoprotein as chiral selector by using a mobile phase consisting of a 10-mM acetate buffer (pH 4.5) and 1-propanol (99:1, v/v). The LC system is coupled to tandem mass spectrometry with an APCI interface in the positive-ion mode. The chromatographed analytes are detected in the selected reaction monitoring mode (SRM). The MS/MS ion transitions monitored are 409 to 238 for amlodipine, and 260 to 116 for S-(-)-propranolol used as internal standard (IS). The method was validated considering different parameters, such as linearity, precision and accuracy. The limit of quantitation was found to be 0.1 ng/ml for each amlodipine enantiomer.     

Simultaneous determination of bisoprolol and hydrochlorothiazide in human plasma by HPLC coupled with tandem mass spectrometry

A sensitive, specific and selective method has been developed for the simultaneous determination of bisoprolol and hydrochlorothiazide in human plasma. The method employed a state of the art LC–MS/MS operated in the positive and negative ionization switching modes. A simple sample preparation step involving protein precipitation with acetonitrile has been optimized; the analytes and the internal standard moxifloxacin were separated on a Purosphere^® STAR C₈ column (125 mm × 4 mm, 5 μm). The mobile phase was an ammonium acetate solution (1 mM) with formic acid (0.2%): methanol and acetonitrile (65:17.5:17.5, v/v/v (%)), the flow rate was set at 0.65 mL/min. Bisoprolol and hydrochlorothiazide were ionized using ESI source prior to detection by Multiple Reaction Monitoring (MRM) mode while monitoring at the following transitions: positive m/z 326 → 116 for bisoprolol, negative m/z 296 → 269 and m/z 296 → 205 for hydrochlorothiazide. Linearity was demonstrated over the concentration range 0.10–30.0 (ng/mL) for bisoprolol and 1.00–80.00 ng/mL for hydrochlorothiazide. The limits of detection were 0.100 (ng/mL) for bisoprolol and 1.00 (ng/mL) for hydrochlorothiazide. The validated method was successfully applied to a pharmacokinetic study of 5 mg bisoprolol fumarate with 12.5 mg hydrochlorothiazide tablet in healthy volunteers.     

Quantitation of entacapone glucuronide in rat plasma by on-line coupled restricted access media column and liquid chromatography–tandem mass spectrometry

A column-switching liquid chromatography–electrospray ionization-tandem mass spectrometric (LC–ESI-MS–MS) method was developed for the direct analysis of entacapone glucuronide in plasma. The plasma samples (5 μl) were injected onto a C₁₈-alkyl-diol silica (ADS) column and the matrix compounds were washed to waste with a mixture of 20 mM ammonium acetate solution at pH 4.0–acetonitrile (97:3). The retained analyte fraction containing (E)- and (Z)-isomers of glucuronides of entacapone and tolcapone glucuronide (internal standard) was backflushed to the analytical C₁₈ column, with a mixture of 20 mM ammonium acetate–acetonitrile (85:15) for the final separation at pH 7.0. The eluate was directed to the mass spectrometer after splitting (1:100). The mass spectrometer was operated in the negative ion mode and the deprotonated molecules [M−H]⁻ were chosen as precursor ions for the analytes and internal standard. Collisionally induced dissociation of [M−H]⁻ in MS–MS resulted in loss of the neutral glucuronide moiety and in the appearance of intensive negatively charged aglycones [M−H−Glu]⁻, which were chosen as the product ions for single reaction monitoring. Quantitative studies showed a wide dynamic range (0.0025–100 μg/ml) with correlation coefficients better than 0.995. The method was repeatable within-day (relative standard deviation, RSD<7%) and between-day (RSD<14%) and the recovery (78–103%) was better than with the traditional, laborious pretreatment method. The use of tandem mass spectrometry permitted low limits of detection (1 ng/ml of entacapone glucuronide). The method was applied for the quantitation of (E)- and (Z)-isomers of entacapone glucuronide in plasma of rats used in absorption studies.     

Simultaneous analysis of chlorpyrifos and cypermethrin in cord blood plasma by online solid-phase extraction coupled with liquid chromatography-heated electrospray ionization tandem mass spectrometry

Chlorpyrifos and cypermethrin are the most used insecticides in Taiwan. Exposure to both pesticides has been associated with reproductive and developmental health effects in humans and animals. This study describes an online solid-phase extraction coupled with liquid chromatography-heated electrospray ionization tandem mass spectrometry (online SPE-LC/HESI/MS/MS) method to analyze chlorpyrifos and cypermethrin in cord blood of pregnant women. Calibration curves showed good linearity (r2 > 0.998) for both pesticides within the range of 0.1-100 ppb. Limits of detection (LODs) were 0.01 and 0.05 ppb and recoveries in cord blood were 97.2 ± 4.8% and 93.5 ± 9.5% for chlorpyrifos and cypermethrin respectively. After analysis of 396 samples, the mean concentrations of chlorpyrifos and cypermethrin were 0.38 and 1.08 ppb respectively. These results demonstrate that LC/HESI/MS/MS is effective for the simultaneous analysis of chlorpyrifos and cypermethrin in cord blood with excellent sensitivity and specificity and may also be effective for high throughput assay in future epidemiology studies.     

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.     

Amino acid analysis by hydrophilic interaction chromatography coupled on-line to electrospray ionization mass spectrometry

Summary. Collagens form a common family of triple-helical proteins classified in 21 types. This unique structure is further stabilized by specific hydroxylation of distinct lysyl and prolyl residues forming 5-hydroxylysine and hydroxyproline (Hyp) isomers, mostly 4-trans and 3-trans-Hyp. The molecular distribution of the Hyp-isomers among the different collagen types is still not well investigated, even though disturbances in the hydroxylation of collagens are likely to be involved in several diseases such as osteoporosis and autoimmune diseases. Here, a new approach to analyze underivatized amino acids by hydrophilic interaction chromatography (HILIC) coupled on-line to electrospray ionization mass spectrometry (ESI-MS) is reported. This method can separate all three studied Hyp-isomers, Ile, and Leu, which are all isobaric, allowing a direct qualitative and quantitative analysis of collagen hydrolysates. The sensitivity and specificity was increased by a neutral loss scan based on the loss of formic acid (46 u).     

Rapid determination of sixteen sulfonylurea herbicides in surface water by solid phase extraction cleanup and ultra-high-pressure liquid chromatography coupled with tandem mass spectrometry

A sensitive and very fast analytical method has been developed for the simultaneous quantification of sixteen sulfonylurea herbicides in surface water. An ultra-high-pressure liquid chromatography coupled with tandem mass spectrometry method with solid phase extraction for sample cleanup has been developed for screening sixteen sulfonylurea herbicides (oxasulfuron, thifensulfuron-methyl, cinosulfuron, metsulfuron methyl, sulfometuron methyl, triasulfuron, rimsulfuron, ethametsulfuron methyl, sulfosulfuron, tribenuron methyl, bensulfuron methyl, iodosulfuron methyl, pyrazosulfuron ethyl, prosulfuron, chlorimuron ethyl, ethoxysulfuron) in water samples simultaneously within 12 min. Water samples were acidified, and the target herbicides were extracted by passing through ProElut C18 extraction cartridges. After drying by nitrogen flow, the cartridges were eluted with elution solvents, and the eluate was then evaporated to dryness, redissolved and analyzed. The mobile phase composed of 0.02% formic acid and acetonitrile using gradient elution. A triple quadrupole mass spectrometer equipped with an electrospray ionization source operated in the positive ion with selective reaction monitoring mode. Each of the analytes in all the samples was monitored using protonated molecule and its two characteristic fragment ions for confirmation. The limits of detection for all analytes were below 1.0 ng/mL, except for sulfosulfuron and prosulfuron, and limits of quantitation were between 1 and 8 ng/mL for this method. Three water types were used for the validation of the method.     

Automated, fast and sensitive quantification of 17 alpha-hydroxy-progesterone, androstenedione and testosterone by tandem mass spectrometry with on-line extraction

Plasma 17 alpha-hydroxyprogesterone (17-OHP), androstenedione and testosterone measurements are important for the diagnosis and monitoring of hyperandrogenic disorders, most importantly for congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency. The reliability of immunoassays has proved questionable especially for newborns and children. In order to reduce the analytical interferences due to cross-reactivity or matrix effects, to improve accuracy and shorten the analysis time, we have developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with atmospheric pressure chemical ionization (APCI) for simultaneous measurement. An on-line extraction cartridge with column-switching technique and liquid chromatography over a Chromolith RP 18 e column allow a rapid and easy quantification. The lowest limit of detection was 0.03-0.06 microg/L. Our method has proved linear up to 250 microg/L (r=0.999). Recoveries (S.D.) of 17-OHP, androstenedione and testosterone in plasma were 100% (5), 102% (2) and 92% (4), respectively. The regression equation for the LC-MS/MS (x) and immunoassay (y) methods for 17-OHP (excluding neonate samples) was y=1.942 x+0.255 nmol/L (r=0.695; n=97). In comparison to our values, the immunoassay generally overestimates steroid concentration. The regression equation for the LC-MS/MS (x) and immunoassay (y) methods for testosterone was y=0.963 x+0.035 nmol/L (r=0.955; n=107). Preliminary reference intervals for children were determined as a function of age and sex. The sensitivity and specificity of the LC-MS/MS method offer advantages over routine immunoassays due to the elimination of interferences especially for newborns, high throughput and short chromatographic run time.     

Determination of the glucocorticoid fluticasone propionate in plasma by automated solid-phase extraction and liquid chromatography–tandem mass spectrometry

A sensitive, robust and high throughput mass spectrometry based method is described for the determination of the glucocorticoid fluticasone propionate in plasma. The method employs solid-phase extraction in 96 well microtitre plate format which has been automated by means of a custom built Zymark robotic system. The extracts are analysed by liquid chromatography–tandem mass spectrometry using thermally and pneumatically assisted electrospray ionisation and selected reaction monitoring. The method is both accurate and precise with both intra- and inter-assay precision (C.V.) of less than <6%. The method provides a lower limit of quantification of 20 pg/ml from 0.5 ml of human plasma, sufficient to monitor systemic concentrations of inhaled fluticasone propionate at therapeutic doses.     

Rapid determination of gemifloxacin in human plasma by high-performance liquid chromatography–tandem mass spectrometry

A method was developed for the determination of gemifloxacin (I) in human plasma using high-performance liquid chromatography–tandem mass spectrometry. Prior to analysis, the protein in plasma samples was precipitated with acetonitrile containing [¹³C²H₃] gemifloxacin (II) to act as an internal standard. The supernatant was injected onto a PLRP-S column without any further clean-up. The mass spectrometer was operated in positive ion mode, employing a heat assisted nebulisation, electrospray interface. Ions were detected in multiple reaction monitoring (MRM) mode. The assay requires 50 μl of plasma and is precise and accurate within the range 10–5000 ng/ml. The average within-run and between-run coefficients of variation were <11% at 10 ng/ml and greater concentrations. The average accuracy of validation standards was generally within ±7% of the nominal concentration. There was no evidence of instability of I in human plasma following three complete freeze–thaw cycles and samples can safely be stored for at least 6 months at −20°C. The method proved very robust and was successfully applied to the analysis of clinical samples from patients dosed with gemifloxacin.     

Development of a novel method for triterpenoidal saponins in rat plasma by solid-phase extraction and high-performance liquid chromatography tandem mass spectrometry

A novel method using high-performance liquid chromatography tandem mass spectrometry (HPLC–MS/MS) has been developed for the quantification of four triterpenoidal saponins (anemoside B4, pulsatilloside B, anemoside A3, and 23-hydroxybetulinic acid) in rat plasma following solid-phase extraction (SPE). The optimized procedure utilized off-line extraction of the analytes from plasma using polymeric (Strata-X) SPE cartridges. Detection and quantitation were performed by MS/MS using electrospray ionization (ESI) and multiple reaction monitoring (MRM) in a novel multiswitching monitoring mode. The analytes and internal standard (scutellarin) were analyzed using a Sapphire C18 column (250 × 4.6 mm, 5 μm) with a linear gradient elution. The mass transition ion pairs of the triterpenoidal saponins were executed as follows: m/z 1219.7/749.4 for anemoside B4, m/z 819.4/347.2 for pulsatilloside B, m/z 749.6/471.2 for anemoside A3, m/z 471.4/471.4 for 23-hydroxybetulinic acid, and m/z 461.1/285.0 for the internal standard. The specificity, linearity, accuracy, precision, recovery, matrix effect, and stabilities were validated for all analytes in the plasma samples. In conclusion, the validation results demonstrate that this method is robust and specific. This validated method is a novel technique for sample preparation and quantitation and was successfully applied to estimate the pharmacokinetics of triterpenoidal saponins.     

Determination of deoxynivalenol-sulfonate (DONS) in cereals by hydrophilic interaction chromatography coupled to tandem mass spectrometry

Deoxynivalenol (DON) is one of most widespread mycotoxins in cereal commodities, and animal feed is prevalently contaminated at high concentrations. This poses a problem in animal nutrition as especially pigs are very sensitive to DON. An effective process for the reduction of the DON concentration is the treatment of contaminated feed with sodium bisulfite (SBS) whereby DON is transformed into DON-sulfonate (DONS). Although the success of this treatment has been confirmed in several feeding studies, it is unexplained if the decrease of DON is accompanied with a coincident increase of DONS. For this reason, we developed a method for the analysis of DONS using hydrophilic interaction chromatography coupled to tandem mass spectrometry. In order to investigate the correlation between DON and DONS concentrations during SBS-treatment, DON-contaminated wheat was treated with SBS and stored for up to 36 days. At defined timepoints of this treatment, samples were analyzed for DON and DONS using stable isotope labeled standards. The preparation, purification, and structure elucidation of DONS, and the HILIC-HPLC-MS/MS method for the analysis of DONS as well as the results of two storage experiments are presented in this paper.     

Determination of palmatine in canine plasma by liquid chromatography-tandem mass spectrometry with solid-phase extraction

A sensitive and specific high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS-MS) method has been developed and validated for the determination of palmatine in canine plasma. Palmatine and jatrorrhizine (internal standard, I.S.) were extracted from plasma samples by solid-phase extraction (SPE) using Oasis HLB cartridges. The chromatographic separation was performed on a Waters XTerra MS C(18) reversed-phase column at 30 degrees C. The gradient mobile phase, delivered at 0.25 mL/min, was composed of a mixture of acetonitrile -0.1% (v/v) acetic acid aqueous solution adjusted to pH 2.8 with triethylamine. Positive electrospray ionization was utilized as the ionization source. Palmatine and the internal standard (I.S.) were determined using multiple reaction monitoring (MRM) of precursor-->product ion transitions at m/z 352-->336 and m/z 338-->322, respectively. The lower limit of quantification (LLOQ) was 0.1 ng/mL using 100 microL plasma samples and the linear calibration range was from 0.1 to 500 ng/mL. The inter-day and intra-day RSDs were lower than 9.9% and the recoveries of palmatine ranged from 87.3 to 100.9%. The mean extraction recoveries of palmatine and the I.S. were 99.2 and 96.8%, respectively. The method has been successfully applied to the pharmacokinetic studies of palmatine in beagle dogs after oral administration and intramuscular injection of palmatine.     

Simultaneous quantitative determination of olmesartan and hydrochlorothiazide in human plasma and urine by liquid chromatography coupled to tandem mass spectrometry

A specific, sensitive and rapid method based on high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) was developed for the simultaneous determination of olmesartan (OLM) and hydrochlorothiazide (HCTZ) in human plasma and urine. Solid-phase extraction (SPE) was used to isolate the analytes from biological matrices followed by injection of the extracts onto a C₁₈ column with isocratic elution. Detection was carried out on a triple quadrupole tandem mass spectrometer in multiple reaction monitoring (MRM) mode using negative electrospray ionization (ESI). The method was validated over the concentration range of 1.00–1000 ng/mL and 5.00–5000 ng/mL for OLM in human plasma and urine as well as 0.500–200 ng/mL and 25.0–25,000 ng/mL for HCTZ in human plasma and urine, respectively. Inter- and intra-run precision of OLM and HCTZ were less than 15% and the accuracy was within 85–115% for both plasma and urine. The average extraction recoveries were 96.6% and 92.7% for OLM, and 87.2% and 72.1% for HCTZ in human plasma and urine, respectively. The linearity, recovery, matrix effect and stability were validated for OLM/HCTZ in human plasma and urine.     

Quantitative determination of plasma vitamin K1 by high-performance liquid chromatography coupled to isotope dilution tandem mass spectrometry

Plasma vitamin K1 (phylloquinone) determination is commonly used for the diagnosis of vitamin K deficiency in patients suffering from lipid malabsorption. Moreover, current evidence that adequate vitamin K intake, and correspondingly adequate plasma vitamin K1 concentration, could also be of importance in relation to bone and brain diseases emphasizes the need to improve the current analytical methods. We developed a liquid chromatography coupled to tandem mass spectrometry method using a stable isotope ring-D4-labeled internal standard of vitamin K1 and operating in the multiple reaction monitoring mode by the selection of a precursor and product ions. The atmospheric pressure chemical ionization (APCI) method was shown to be more sensitive than electrospray ionization. After a single-step extraction with cyclohexane, chromatographic separation was performed on a C18 column with an isocratic mobile phase. The linearity was up to 5400 ng/L, and the limit of detection was 14 ng/L. Intra- and interrun precision were 2.4% and 8.3%, respectively, for the lower limit of the reference range. Recovery was better than 98%. The method is simple and reliable, allowing accurate vitamin K1 measurement in plasma samples from healthy subjects and patients suffering from vitamin K deficiency.