Determination of misoprostol acid in human plasma by liquid chromatography coupled to tandem mass spectrometry

A rapid and sensitive liquid chromatographic/tandem mass spectrometric method for determination of misoprostol acid, the active metabolite of misoprostol, was developed and validated. Following liquid-liquid extraction, the analytes were separated using an isocratic mobile phase on a C(18) column. An API 4000 tandem mass spectrometer equipped with Turbo IonSpray ionization source was used as detector and was operated in the negative ion mode. Multiple reaction monitoring using the precursor to product ion combinations of m/z 367-249 and 296-269 was performed to quantify misoprostol acid and the internal standard hydrochlorothiazide, respectively. The method was linear in the concentration range of 10.0-3000 pg mL(-1) using 200 microL plasma. The lower limit of quantification was 10.0 pg mL(-1). The intra- and inter-day relative standard deviation over the entire concentration range was less than 8.3%. Accuracy determined at three concentrations (25.0, 200 and 2700 pg mL(-1) for misoprostol acid) ranged from -0.5 to 1.2% in terms of relative error. Each plasma sample was chromatographed within 3.5 min. The method was successfully used in a pharmacokinetic study of misoprostol in human plasma after an oral administration of 0.6 mg misoprostol.     

Determination of serum methylmalonic acid by alkylative extraction and liquid chromatography coupled to tandem mass spectrometry

Despite the new advances in bioanalytical techniques, the analysis of low-molecular-weight organic acids in complex matrices is still a challenge. Although new strategies applying liquid chromatography-tandem mass spectrometry (LC-MS/MS) seem to be promising, sample preparation methodologies hamper its application in most clinical laboratories. The quantitation of methylmalonic acid (MMA) in biological matrices is an emblematic example due to its low concentration, the need for derivatization to increase its molecular weight, and the presence of the physiologically more abundant isomer succinic acid. Here we present a new strategy for rapid and sensitive MMA quantitation by combining alkylative extraction and LC-MS/MS. Alkylative extraction conditions were optimized to allow endogenous detection of MMA using only 50 μL of serum with a short sample preparation procedure. The formation of a unique ion from the MMA dipentafluorobenzyl derivative in negative atmospheric pressure chemical ionization (APCI) allowed its detection with high sensitivity and with no interference from succinic acid, a more abundant physiologically present isomer.     

Determination of LSD and N-demethyl-LSD in urine by liquid chromatography coupled to electrospray ionization mass spectrometry

A sensitive and highly specific method for the determination of LSD and N-demethyl-LSD in urine, using combined liquid chromatography and mass spectrometry (LC-MS) with electrospray ionization, has been developed. Extrelut-3 extraction cartridges were used for a basic sample clean-up. Elution was obtained by toluene-diethyl ether (60:40, v/v). A Nucleosil C₁₈ (150×1 mm I.D.) reversed-phase column was used for the chromatographic separation, together with a mixture of 2 mM ammonium formate buffer (pH 3) and acetonitrile (70:30, v/v) as mobile phase. Recoveries were 93 and 80%, detection limits 0.025 and 0.035 ng/ml for LSD and N-demethyl-LSD, respectively. Intra-assay precision, studied at four concentrations, was better than 9% at the ng/ml range and better than 14% at 0.10 ng/ml for both compounds. Limits of quantitation were 0.05 and 0.10 ng/ml for LSD and N-demethyl-LSD, respectively. Reproducibility was good and linearity excellent for LSD in the range from 0.05 to 20 ng/ml (r>0.9999, N=7).     

Simultaneous analysis of cyclophosphamide, doxorubicin and doxorubicinol by liquid chromatography coupled to tandem mass spectrometry

A method for the simultaneous determination of cyclophosphamide (CP), doxorubicin (dox), and doxorubicinol (dol) was developed and validated to analyze 400 microL of plasma from patients receiving chemotherapeutic treatment with CP and dox. Final calibration ranges for the analytes were 0.440-60.0 microg/mL for cyclophosphamide, 7.20-984 ng/mL for dox and 3.04-104 ng/mL for dol. The samples were prepared using solid phase extraction and analyzed using a gradient separation over a Waters Symmetry C18, 2.1 by 30 mm (Milford, MA) column. Detection was achieved in positive mixed reaction monitoring mode on a triple quadrupole mass spectrometer.     

Felodipine quantification in human plasma by high-performance liquid chromatography coupled to tandem mass spectrometry

A rapid, sensitive, robust and specific method was developed for the determination and quantitation of felodipine, in human blood plasma by liquid chromatography coupled with tandem mass spectrometry using nimodipine as internal standard. Felodipine was extracted from 0.5 mL human plasma by use of a liquid/liquid procedure using diethyl ether/hexane (80/20, v/v) as eluent. The method included a chromatographic run of 5 min using a C(18) analytical column (100 mm x 4.6 mm i.d.) and the calibration curve was linear over the range from 0.02 to 10 ng mL(-1) (r(2) > 0.994). The between-run precision, determined as relative standard deviation of replicate quality controls, was 5.7% (0.06 ng mL(-1)), 7.1% (0.6 ng mL(-1)) and 6.8% (7.5 ng mL(-1)). The between-run accuracy was +/- 0.0, 2.1 and 3.1% for the above-mentioned concentrations, respectively.     

Glycolate and 2-phosphoglycolate content of tissues measured by ion chromatography coupled to mass spectrometry

Glycolate and 2-phosphoglycolate (PG) are 2-carbon monocarboxylic acids with ill-defined metabolic roles. Their concentrations have not yet been described in tissues apart from body fluids and erythrocytes. We describe the use of ion chromatography coupled with mass spectrometry (IC-MS) to quantify levels of glycolate and PG in tissue. Sample preparation and analysis can be performed within an hour. Low concentrations of glycolate (12-48 nmol/g) and PG (4-17 nmol/g) were detected in all tissues. The availability of this IC-MS assay will facilitate investigations of the origin, function, and metabolism of glycolate and PG in tissues.     

Untargeted large-scale plant metabolomics using liquid chromatography coupled to mass spectrometry

Untargeted metabolomics aims to gather information on as many metabolites as possible in biological systems by taking into account all information present in the data sets. Here we describe a detailed protocol for large-scale untargeted metabolomics of plant tissues, based on reversed phase liquid chromatography coupled to high-resolution mass spectrometry (LC-QTOF MS) of aqueous methanol extracts. Dedicated software, MetAlign, is used for automated baseline correction and alignment of all extracted mass peaks across all samples, producing detailed information on the relative abundance of thousands of mass signals representing hundreds of metabolites. Subsequent statistics and bioinformatics tools can be used to provide a detailed view on the differences and similarities between (groups of) samples or to link metabolomics data to other systems biology information, genetic markers and/or specific quality parameters. The complete procedure from metabolite extraction to assembly of a data matrix with aligned mass signal intensities takes about 6 days for 50 samples.     

Quantitative bioanalysis of peptides by liquid chromatography coupled to (tandem) mass spectrometry

With the growing interest for peptides and proteins in different kinds of fields, e.g. pharmacy, clinical diagnostics or food industry, the quantification of these compounds is becoming more and more important. Quantitative analysis of these analytes in biological matrices, however, remains a challenging task, due to the complexity of both the matrix and the analytical characteristics of these large bio-molecules. Liquid chromatography coupled to (tandem) mass spectrometry (LC-MS or LC-MS/MS) is the preferred analytical technique for peptide analysis as it allows very selective and sensitive measurements. This article summarizes the numerous published LC-MS applications for the quantification of peptides in biological matrices and discusses all different issues herewith concerned. This includes chromatographic aspects as the selection and effects of mobile and stationary phase, flow rate and temperature, as well as mass spectrometric characteristics such as ionization and detection modes, collision-induced dissociation of peptides and factors influencing the mass spectrometric response. For both techniques the main properties of all described methods have been listed, creating a comprehensive overview with the peptide analytes divided into different classes. Likewise, all other issues concerned with quantitative bioanalysis have been evaluated in detail, including extensive consideration of several different applied sample pre-treatment techniques and reflection of subjects as the choice for an internal standard and assay validation. Furthermore, several issues which are of particular interest for the quantitative bioanalysis of peptide compounds like peptide adsorption and degradation have been regarded.     

Determination of ritodrine in human plasma by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

A simple and sensitive HPLC/MS/MS method was developed and evaluated to determine the concentration of ritodrine (RTD) in human plasma. Liquid-liquid extraction with ethyl acetate was employed as the sample preparation method. The structural analogue salbutamol was selected as the internal standard (IS). The liquid chromatography was performed on a Hanbon Sci. & Tech. Lichrospher CN (150 mm x 4.6 mm, i.d., 5 microm) column (Hanbon, China) at 20 degrees C. A mixture of 0.03% acetic acid and methanol (50:50, v/v) was used as isocratic mobile phase to give the retention time 3.60 min for ritodrine and 2.94 min for salbutamol. Selected reaction monitoring (SRM) in positive ionization mode was employed for mass detection. The calibration functions were linear over the concentration range 0.39-100 ng mL(-1). The intra- and inter-day precision of the method were less than 15%. The lower limit of quantification was 0.39 ng mL(-1). The method had been found to be suitable for application to a pharmacokinetic study after oral administration of 20mg ritodrine hydrochloride tablet to 18 healthy female volunteers. The half-life is 2.54+/-0.67 h.     

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.     

Separation and determination of Se-compounds by liquid chromatography coupled with electrospray mass spectrometry

A method for Selenocystine and Selenomethionine determination by LC–ES–MS was developed in this work. The mass spectrometer was used in a positive mode and the m/z used for the identification of Selenomethionine and Selenocystine were 198.35 and 337.15, respectively.The selenium species were separated using a LC system. A silica chromatographic column (ZORBAX Eclipse XDB-C₈ of 50 mm length and 2.1 mm internal diameter (particle size 3.5 μm)) was used. The separation was realised in isocratic mode, using methanol:water (1:1) with 1% of acetic acid and a flow rate of 200 μL min⁻¹. The developed method was precise (RSD of 4.5% and 3.9% for Selenomethionine and Selenocystine, respectively) and sensible (limit of detection (LOD) 0.06 and 0.99 mg L⁻¹ for selenomethionine and selenocystine, respectively).     

Determination of quinapril and quinaprilat by high-performance liquid chromatography with radiochemical detection,coupled to liquid scintillation counting spectrometry

Quinapril and quinaprilat concentrations were determined in perfusate, urine, and perfusate ultrafiltrate using a specific and sensitive reversed-phase high-performance liquid chromatographic procedure with radiochemical detection, coupled to liquid scintillation counting spectrometry. Quinapril and quinaprilat were measured in perfusate and urine after pretreatment with acetonitrile and subsequent centrifugation. Perfusate ultrafiltrate was used as collected. Two quinapril diketopiperazine metabolites, PD 109488 and PD 113413, were separated chromatographically from quinapril, quinaprilat, and from each other. Assay performance for quinapril and quinaprilat was assessed by examining precision and accuracy of the assay over four days. Using a 100-μl sample volume, the limit of quantitation for both ³H-quinapril and ³H-quinaprilat (sp. act. ≈ 2.0 μCi/μg) was 1 ng/ml.     

High-performance liquid chromatography coupled with negative ion tandem mass spectrometry for determination of pravastatin in human plasma

A new method, using high-performance liquid chromatography/ion electrospray (negative ion) mass spectrometry, has been developed for the determination of a hydrophilic liver-specific inhibitor of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase, pravastatin in human plasma. In this method, plasma samples were prepared by a solid-phase extraction on C(18) Bond Elut cartridge. Chromatography was carried out with a Zorbax C(8) column. Simple isocratic chromatography conditions were used. The method has been validated in a linear range of 0.25-300 ng/ml with a coefficient of variation of 0.6-3.4%. The overall recovery was 90.5% for pravastatin and 90.8% for the internal standard beta-hydroxy-lovastatin. The method is simple and reliable with a total run time of less than 2 min.     

Characterization of metallothionein isoforms from rabbit liver by liquid chromatography coupled to electrospray mass spectrometry

Metallothioneins (MTs), a group of low molecular weight proteins found in practically all life forms, are characterized by high sulfur content and an affinity for metal ions. At acidic pH, MTs show metal depletion, leading to apothioneins. In the work described here, in order to optimize the separation of rabbit liver apothioneins using liquid chromatography (LC) with UV detection, the proportion of the organic modifier of the mobile phase was optimized by establishing relationships between Reichardt's E(N)(T) scale of solvent polarity and the chromatographic retention measured by the capacity factor, k. Additionally, such optimum separations were carried out in a LC-electrospray mass spectrometry (ES-MS) coupled system allowing the identification and characterization of the different rabbit liver apo-MT-forms. In this way, electrospray ionization mass spectrometry offers great possibilities aiming at a better understanding of metallothionein polymorphism.     

Determination of mycotoxins in bovine milk by liquid chromatography tandem mass spectrometry

Liquid chromatographic/tandem mass spectrometric methods using pneumatically assisted electrospray ionisation (LC-ESI-MS/MS) was developed for determination of 18 mycotoxins and metabolites-ochratoxin A, zearalenone, alpha-zearalenol, beta-zearalenol, alpha-zearalanol (zeranol), beta-zearalanol (taleranol), fumonisin B1, fumonisin B2, T-2 toxin, HT-2 toxin, T-2 triol, diacetoxyscirpenol (DAS), 15-monoacetoxyscirpenol (MAS), deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-AcDON), 15-acetyldeoxynivalenol (15-AcDON), deepoxy-deoxynivalenol (DOM-1) and aflatoxin M1--in milk. The mycotoxins were extracted and cleaned up simultaneously. Extraction and removal of lipophilic compounds was performed at pH 2 using a two-phase mixture of acetonitrile and hexane. The acetonitrile concentration of the aqueous phase was reduced and the pH was adjusted to 8.5 before clean up by solid phase extraction (SPE) on Oasis HLB. The toxins DON, DOM-1, 3-AcDON, 15-AcDON, ochratoxin A, zearalenone, alpha-zearalenol, beta-zearalenol, alpha-zearalanol and beta-zearalanol were detected in negative ion mode after separation on a Hypersil ENV analytical column, while the toxins T-2 toxin, HT-2 toxin, T-2 triol, DAS, MAS, fumonisin B1, fumonisin B2 and aflatoxin M1 were detected in positive ion mode after separation on a Luna C18 column. Two transition products were monitored for each compound. The extraction and SPE conditions were optimised to obtain maximum recovery and minimum signal suppression/enhancement. The detection capabilities related to the transition products of lowest abundance were in the range 0.020-0.15 microg/l. The mean true recoveries were in the range 76-108% at levels of 0.2-10 microg/l.     

Determination of the glucocorticoids prednisone, prednisolone, dexamethasone, and cortisol in human serum using liquid chromatography coupled to tandem mass spectrometry

Glucocorticoids are an important component of immunosuppressive therapy for solid organ transplantation. A method to quantitate prednisone, prednisolone, dexamethasone and cortisol in human serum has been developed. Analysis is performed utilizing reversed-phase liquid chromatography coupled to tandem mass spectrometry. The method was validated to a lower limit of quantitation of 5.4 ng/ml for prednisone and cortisol, and 10.7 ng/ml for dexamethasone and prednisolone, with error below 7% at the lower limits. The between-day relative standard deviations ranged 2.9-7.1%. Comparison of cortisol analysis to an established method using clinical samples yielded differences below 15% for 26 of 28 determinations.     

Structural characterization of intact, branched oligosaccharides by high performance liquid chromatography and liquid secondary ion mass spectrometry

We report results of a mass-spectrometric-based strategy for determining the detailed structural features of N-linked oligosaccharides from glycoproteins. The method was used to characterize a series of intact, high mannose oligosaccharides isolated from human immunoglobulin M (IgM). The IgM was purified from a patient with Waldenstrom's macroglobulinemia. The strategy included releasing the oligosaccharides by digestion of the purified glycoprotein with endoglycosidase H, separating the released oligosaccharides by high resolution gel filtration, and derivatizing the resulting reducing termini with the uv-absorbing moiety, ethyl p-aminobenzoate. This particular derivative facilitates HPLC detection and provides centers for protonation and deprotonation enhancing liquid secondary ion mass spectra. Positive and negative ion spectra contained molecular species of similar abundance. However, fragment ion peaks yielding sequence information were significantly more prominent in the negative ion mass spectra. Furthermore, it was obvious that the fragmentation patterns differed substantially for linear and branched oligomers. For linear oligosaccharides, a smooth envelope of fragment ions was observed; from low to high mass there was an ordered decrease in ion abundance from both the reducing and nonreducing termini. This pattern of fragment ions was not observed for branched oligosaccharides since in these cases fragments at certain masses could not arise by single bond cleavages. Therefore, these fragments were either significantly reduced in abundance or absent as compared with identical fragments formed from linear molecules. Importantly, 200 pmol of an oligosaccharide could be derivatized, separated, and detected by mass spectrometry, allowing identification of previously unreported minor components of the IgM oligosaccharides. Therefore, this experimental strategy is particularly useful for the purification and detailed structural characterization of low abundance oligosaccharides isolated from heterogeneous biological samples.