Resolution of triacylglycerol positional isomers by reversed-phase high-performance liquid chromatography

The ability of reversed-phase high-performance liquid chromatography (RP-HPLC) to separate some positionally isomeric disaturated and monounsaturated triacylglycerols (TAGs) as intact species is demonstrated for the first time. Mobile phases of acetonitrile modified with methanol, ethanol, 2-propanol, 1-propanol, 1-butanol, acetone, or dichloromethane were tested for the separation of POP-PPO, PLP-PPL, PEP-PPE, and PDP-PPD (P-palmitic, O-oleic, L-linoleic, E-eicosapentaenoic, D-docosahexaenoic acid residue) on a single RP-HPLC column. The resolution improved with increasing number of double bonds in the acyl residues. While POP and PPO were only partially resolved, PDP and PPD were fully separated with all tested mobile phases, except those containing methanol. Also separated were the four TAGs having the same equivalent carbon number (ECN = 42), PEP, PPE, PDP, and PPD, on a single RP-HPLC column with mobile phase acetonitrile-2-propanol (70:30, v/v) at 0.8 mL/min. In all cases the isomer with the unsaturated acyl residue in either 1- or 3-position was retained more strongly than the respective 2-isomer.     

Development and validation of a sensitive LC-MS/MS method with electrospray ionization for quantitation of pramipexole in human plasma: application to a clinical pharmacokinetic study

A highly sensitive and specific LC-MS/MS method has been developed and validated for the estimation of pramipexole (PPX) with 500 microL human plasma using memantine as an internal standard (IS). The API-4000 was operated under multiple-reaction monitoring mode (MRM) using the electrospray ionization technique. Solid-phase extraction was used to extract PPX and IS from human plasma. The resolution of peaks was achieved with 0.01 m ammonium acetate buffer (pH 4.4):acetonitrile (30:70, v/v) on a Discovery CN column. The total chromatographic run time was 3.0 min and the elution of PPX and IS occurred at approximately 2.32 and 2.52, respectively. The MS/MS ion transitions monitored were 212.10 --> 153.10 for PPX and 180.20 --> 107.30 for IS. The method was proved to be accurate and precise at linearity range of 20-3540 pg/mL with a correlation coefficient (r) of > or =0.999. The intra- and inter-day precision and accuracy values found to be within the assay variability limits as per the FDA guidelines. The developed assay method was applied to a pharmacokinetic study in human volunteers following oral administration of 0.25 mg PPX tablet.     

Reversed-phase capillary electrochromatography for the simultaneous determination of acetylsalicylic acid, paracetamol, and caffeine in analgesic tablets

The separation and simultaneous determination of caffeine, paracetamol, and acetylsalicylic acid in two analgesic tablet formulations was investigated by capillary electrochromatography (CEC). The effect of mobile phase composition on the separation and peak efficiency of the three analytes was studied and evaluated; in particular, the influence of buffer type, buffer pH, and acetonitrile content of the mobile phase was investigated. The analyses were carried out under optimized separation conditions, using a full-packed silica capillary (75 microm ID; 30.0 cm and 21.5 cm total and effective lengths, respectively) with a 5 microm C8 stationary phase. A mixture of 25 mM ammonium formate at pH 3.0 and acetonitrile (30:70 v/v) was used as the mobile phase. UV detection was at 210 nm. Good linearity was found in the range of 50-200, 20-160, and 4-20 microg/mL for acetylsalicylic acid (r2=0.9988), paracetamol (r2=0.9990) and caffeine (r2=0.9990), respectively. Intermediate precision (RSD interday) as low as 0.1-0.8% was found for retention times, while the RSD values for the peak area ratios (Aanalyte/AIS) were in the range of 1.9-2.9%. The optimized CEC method was applied to the analysis of the studied compounds present in commercial tablets.     

Use of different stationary phases for separation of isoniazid, its metabolites and vitamin B6 forms

The ability of different stationary phases developed for the analysis of polar compounds (ZIC-HILIC, ZIC-pHILIC and Zorbax SB-Aq) to separate isoniazid, its metabolites (acetylisonazid, pyridoxal isonicotinoyl hydrazone, pyridoxal isonicotinoyl hydrazone 5-phosphate), pyridoxine, pyridoxal and pyridoxal 5-phosphate under MS compatible conditions was systematically investigated using HPLC-UV. The mobile phase strength, pH and buffer concentration were modified to assess their impact on the retention of these compounds. The best available separation of the compounds was achieved using 1 mM ammonium formate (pH≈6) and ACN (20:80, v/v) on ZIC-HILIC and employing 5 mM ammonium formate (pH 3.0) and ACN (40:60, v/v) on ZIC-pHILIC. A gradient profile using 0.5 mM ammonium formate (pH≈6) and MeOH (0-12 min: 10% MeOH, 12-15 min: 10-50% MeOH, 15-35 min: 50% MeOH, 35.0-35.2 min: 50-10% MeOH, 35.2-45.0 min: 10% MeOH) provided the best separation of the compounds on Zorbax SB-Aq. Subsequent LC-MS analysis demonstrated that ZIC-HILIC is useful for the analysis of pyridoxine, pyridoxal and pyridoxal isonicotinoyl hydrazone. However, the chromatographic conditions developed for the analysis of the compounds on Zorbax SB-Aq are capable of achieving the best separation of all compounds in this study with the higher sensitivity for most of the analytes.     

Simultaneous quantitation of d7-nefazodone, nefazodone, d7-hydroxynefazodone, hydroxynefazodone, m-chlorophenylpiperazine and triazole-dione in human plasma by liquid chromatographic-mass spectrometry

A rapid and sensitive LC-MS assay was developed and validated for the simultaneous determination of d7-nefazodone (d7-NEF), nefazodone (NEF), d7-hydroxynefazodone (d7-OH-NEF), hydroxynefazodone (OH-NEF), m-chlorophenylpiperazine (mCPP), and triazole-dione (Dione) in human plasma using trazodone (TRZ) as the internal standard (IS). A 0.1 mL aliquot of the plasma sample was precipitated with 0.1 mL of acetonitrile and vortexed for 2 min. After centrifugation, 50 microL of supernatant was mixed with 100 microL of 10 mM ammonium formate (pH = 4.0), and a 50 microL aliquot was injected onto a BDS Hypersil C18 column at a flow rate of 0.3 mL/min. The mobile phase consisting of 10 mM ammonium formate (pH = 4) and acetonitrile, 55:45 v/v, was used in an isocratic system. The mass spectrometer was programmed to admit the protonated molecules at m/z 477.2 (d7-NEF), 493.3 (d7-OH-NEF), 197.0 (mCPP), 372.0 (IS), 470.4 (NEF), 458.0 (Dione) and 486.2 (OH-NEF). Standard curves were linear (r(2) >/= 0.994) over the concentration range of 4-1000 ng/mL for Dione and 2-500 ng/mL for all other analytes. The lowest standard concentrations were the lower limits of quantitation for each analyte. The mean predicted quality control concentrations for all analytes deviated by less than 14.3% from the corresponding nominal values; the intra-assay and inter-assay precisions of the assay for all analytes were within 10.5% relative standard deviation. All analytes including the internal standard were stable in the injection solvent at room temperature for at least 24 h. The extraction recovery of the various analytes ranged from 79.2 to 109.1%. The validated assay was applied to the analysis of clinical samples obtained from a human subject who simultaneously received d7-NEF and NEF orally.     

Regioselective separation of isomeric triacylglycerols by reversed-phase high-performance liquid chromatography: stationary phase and mobile phase effects

Complete regioselective separation of five pairs of isomeric dipalmitoyl polyalkenoyl glycerols with two to six double bonds in the unsaturated acyl residues has been achieved by RP-HPLC on a single ODS column. Four ODS columns with stationary phases containing different percentages of free silanol groups have been tested. Binary mobile phases of ACN admixed with dichloromethane, tetrahydrofuran, 1-propanol, 2-propanol, ethanol, or acetone have been examined. The choice of modifier depended on the nature of the stationary phase. The more polar solvents were better suited for stationary phases with higher percentage of free silanol groups. Isomeric species were eluted according to chain length, number of double bonds, and the position of the unsaturated acyl chain in the glycerol molecule. Retention increases in the order 20:5 < 22:6 < 18:3 < 20:4 < 18:2. Within each isomeric pair, the species with unsaturated acyl chain occupying either the sn-1- or the 3-position were retained preferentially. Complete simultaneous regioselective separation of 10 isomeric triacylglycerols in a single isocratic run on a single ODS column was demonstrated.     

Utility of nonaqueous capillary electrophoresis for the determination of lidocaine and its metabolites in human plasma: a comparison of ultraviolet and mass spectrometric detection

A nonaqueous capillary electrophoresis/electrospray mass spectrometry method for the separation of lidocaine (LID) and two of its metabolites, monoethylglycinexylidide (MEGX) and glycinexylidide (GX), has been developed. The separation medium was: 70 mM ammonium formate and 2.0 M formic acid in acetonitrile/methanol (60:40 v/v). With a sheath liquid of methanol/water (80:20 v/v) containing 2% formic acid and positive ion detection, reproducible determinations (8-11% relative standard deviation (RSD)) of lidocaine and its metabolites were performed in spiked human plasma. The limits of detection (LODs) were between 69.1 and 337 nM. The influences of sheath liquid composition, nebulizing gas pressure and drying gas temperature on the separation were examined.     

Analysis of phospholipids by NACE with on-line ESI-MS

A hyphenated method of nonaqueous capillary electrophoresis coupled to electrospray ionization mass spectrometry (NACE-ESI-MS) is described for the simultaneous analysis of phospholipids. The best results were obtained with a mixed solution of methanol/ACN (40:60 v/v) containing 20 mM ammonium acetate and 0.5% acetic acid, under the applied voltage of 30 kV and capillary temperature of 25 degrees C. ESI-MS measurements were performed in the negative mode with methanol/ACN (40:60 v/v) containing 50 mM ammonium acetate as sheath liquid at a flow rate of 2 microL/min. Different phospholipid classes have been successfully separated within 16 min, and the molecular species of every single class have been identified by using MS(2) or MS(3), which generates characteristic fragments through CID. The developed method has been applied to analyze the phospholipids extracted from rat peritoneal surface and the molecular species of phospholipid classes are presented.     

反相高效液相色谱法测定罐头食品中乙二胺四乙酸的残留量

 将罐头食品捣碎后用水稀释,加氯化铜溶液和抗坏血酸,用水定容,过0.45 μm滤膜后立即供液相色谱分析,在254 nm波长处测定。色谱柱：Hypersil ODS(125 mm×4 mm i.d., 5 μm);流动相：水-甲醇（体积比为80∶20）,含有20 mmol/L四丁基溴化铵,0.03 mol/L 醋酸钠缓冲液（pH 4） ,流速：0.8 mL/min;进样量：20 μL。EDTA在10 mg/L～400 mg/L范围内呈线性。方法回收率为95.5%～98.9%,其RSD为0.82%～1.32%。     

A simple and sensitive assay for the quantitative analysis of paclitaxel and metabolites in human plasma using liquid chromatography/tandem mass spectrometry

A sensitive and specific LC-MS/MS assay for the determination of paclitaxel and its 3'p- and 6-alpha-hydroxy metabolites is presented. A 200 microL plasma aliquot was spiked with a 13C6-labeled paclitaxel internal standard and extracted with 1.0 mL tert-butylmethylether. Dried extracts were reconstituted in 0.1 M ammonium acetate-acetonitrile (1:1, v/v) and 25 microL volumes were injected onto the HPLC system. Separation was performed on a 150 x 2.1 mm C18 column using an alkaline eluent (10 mm ammonium hydroxide-methanol, 30:70, v/v). Detection was performed by positive ion electrospray followed by tandem mass spectrometry. The assay quantifies a range for paclitaxel from 0.25 to 1000 ng/mL and metabolites from 0.25 to 100 ng/mL using 200 microL human plasma samples. Validation results demonstrate that paclitaxel and metabolite concentrations can be accurately and precisely quantified in human plasma. This assay is now used to support clinical pharmacologic studies with paclitaxel.     

A new method for screening and determination of diuretics by on-line CE-ESI-MS

A rapid, high-resolution and effective new method for analyzing 12 diuretics by CE-ESI-MS was established in this paper. Ten diuretics (except two neutral compounds) could be fast separated by CE with a DAD at 214 nm with a 20 kV voltage within 6 min, using a 50 microm id and 48.5 cm effective length uncoated fused-silica capillary in a 40 mM ammonium formate buffer (pH 9.40). CE was coupled to the mass spectrometer applying an orthogonal electrospray interface with a triple-tube sheath liquid arrangement. The sheath liquid was composed of isopropanol-water (1:1 v/v) containing 30 mM acetic acid with a flow rate of 4 microL/min. Mass spectrum was employed in the positive mode and both full scan mode and SIM scan mode were utilized. All 12 diuretics could be detected and confirmed by MS in a single analysis. Under optimized conditions, LODs for the 12 diuretics were in the range of 0.13-2.7 micromol/L at an S/N of 3, and the correlation coefficients R(2 )were between 0.9921 and 0.9978. The RDSs (n = 5) of the method was 0.24-0.94 % for migration times and 1.6-8.8 % for peak areas. The recoveries of spiked samples of 12 diuretics were between 72.4% and 118%. The real urine samples were injected directly for analysis, with only simple filtration through a 0.22 microm membrane filter in order to remove solid particles, which may cause capillary blockage. Based on the migration times and characteristic ions, the diuretics in urine samples were detected successfully. This CE-ESI-MS method for analyzing diuretics will hopefully be applied to doping control.     

Enantioseparation of tryptophan and its unnatural derivatives by nano‐LC on CSP‐teicoplanin silica based

This work deals with the potentiality of nano liquid chromatography (Nano‐LC) for the chiral separation of racemic mixture of tryptophan and some selected derivatives by using 100 µm i.d. fused silica capillary packed with teicoplanin bonded to 5 µm diol silica stationary phase. The experiments were carried out by using a cheap and laboratory‐assembled nano‐LC–UV system. Elution was done in an isocratic mode using a polar organic mobile phase. In order to find the optimum chiral separation of the studied enantiomers, some chromatographic experimental parameters were systematically studied and optimized. Among them, mobile phase composition, namely organic modifier type and concentration, buffer type and pH and aqueous content and sample solvent dilution on retention time, retention factor and enantioresolution factor were studied. Baseline enantioresolution and good peak shape was achieved utilizing the mobile phase containing 40 mM ammonium formate at pH pH 2.5 in ACN/water/acetone (60:30:10, v/v/v) at 520 nL/min in less than 8 min analysis time.     

Validation and application of a high-performance liquid chromatography-tandem mass spectrometric method for simultaneous quantification of lopinavir and ritonavir in human plasma using semi-automated 96-well liquid-liquid extraction

Kaletra is an important antiretroviral drug, which has been developed by Abbott Laboratories. It is composed of lopinavir (low-pin-a-veer) and ritonavir (ri-toe-na-veer). Both have been proved to be human immunodeficiency virus (HIV) protease inhibitors and have substantially reduced the morbidity and mortality associated with HIV-1 infection. We have developed and validated an assay, using liquid chromatography coupled with atmospheric pressure chemical ionization tandem mass spectrometry (LC/MS/MS), for the routine quantification of lopinavir and ritonavir in human plasma, in which lopinavir and ritonavir can be simultaneously analyzed with high throughput. The sample preparation consisted of liquid-liquid extraction with a mixture of hexane: ethyl acetate (1:1, v/v), using 100 microL of plasma. Chromatographic separation was performed on a Waters Symmetry C(18) column (150 mm x 3.9 mm, particle size 5 microm) with reverse-phase isocratic using mobile phase of 70:30 (v/v) acetonitrile: 2 mM ammonium acetate aqueous solution containing 0.01% formic acid (v/v) at a flow rate of 1.0 mL/min. A Waters symmetry C(18) guard column (20 mm x 3.9 mm, particle size 5 microm) was connected prior to the analytical column, and a guard column back wash was performed to reduce the analytical column contamination using a mixture of tetrahydrofuran (THF), methanol and water (45:45:10, v/v/v). The analytical run was 4 min. The use of a 96-well plate autosampler allowed a batch size up to 73 study samples. A triple-quadrupole mass spectrometer was operated in a positive ion mode and multiple reaction monitoring (MRM) was used for drug quantification. The method was validated over the concentration ranges of 19-5,300 ng/mL for lopinavir and 11-3,100 ng/mL for ritonavir. A-86093 was used as an internal standard (I.S.). The relative standard deviation (RSD) were <6% for both lopinavir and ritonavir. Mean accuracies were between the designed limits (+/-15%). The robust and rapid LC/MS/MS assay has been successfully applied for routine assay to support bioavailability, bioequivalence, and pharmacokinetics studies.     

Optimization of capillary electrophoretic-electrospray ionization-mass spectrometric analysis of catecholamines

The capillary electrophoretic-mass spectrometric analysis (CE-MS) of catecholamines was optimized with coaxial sheath flow interface and electrospray ionization (ESI). The parameters studied included the sheath liquid composition and its flow rate, separation conditions in ammonium acetate buffer together with the ESI and cone voltages as mass spectrometric parameters. In addition, the effect of ESI voltage on injection as well as the siphoning effect were considered. The optimized conditions were a sheath liquid composition of methanol-water (80:20 v/v) with 0.5% acetic acid, with a flow rate of 6 microL/min. The capillary electrophoretic separation parameters were optimized with 50 mM ammonium acetate buffer, pH 4.0, to +25 kV separation voltage together with a pressure of 0.1 psi. The most intensive signals were obtained with an ESI voltage of +4.0 kV and a cone voltage of +20 V. The nonactive ESI voltage during injection as well as avoidance of the siphoning effect increased the sensitivity of the MS detection considerably. The use of ammonium hydroxide as the CE capillary conditioning solution instead of sodium hydroxide did not affect the CE-MS performance, but allowed the conditioning of the capillary between analyses to be performed in the MS without contaminating the ion source.     

Multielement electrothermal atomic absorption spectrometry: A study on direct and simultaneous determination of chromium and manganese in urine

A study was carried out on the direct determination of Cr and Mn in urine using simultaneous atomic absorption spectrometry (SIMAAS). The heating program conditions, the absorbance signal profiles, the influence of different chemical modifiers, and the urine sample volume delivery into the tube were optimized to perform the calibration with aqueous solutions. Among several chemical modifiers tested, the best recovery and repeatability results were obtained for 3 microg Mg(NO3)2. On using this modifier, the pyrolysis and atomization temperatures for simultaneous determination of Cr and Mn were 1,300 degrees C and 2,500 degrees C, respectively. Urine samples were diluted (1+1) with 2.0% (v/v) HNO3 + 0.05% (w/v) Triton X-100 prepared in high purity water. A 20-microL aliquot of analytical solution and 10 microL of chemical modifier solution were delivered to the graphite tube. The characteristic masses were 7.8 pg for Cr (RSD=4.0%) and 4.6 pg for Mn (RSD=2.6%). The limits of detection were 0.08 microg L(-1) (n=20, 3s) for Cr and 0.16 microg L(-1) (n=20, 3s) for Mn. Recovery studies for 1.0 or 2.5 microg L(-1) of Cr and Mn added to different urine samples showed acceptable results for Cr (100%, RSD=14%) and Mn (88%, RSD=5.6%).     

Direct injection of plasma to determine pseudoephedrine by high performance liquid chromatography with column switching

An HPLC method has been developed for the determination of pseudoephedrine in plasma using column switching. Preparation of the sample was simple in that only 1000 microL of water was added to 200 microL of plasma before injection. A 900 microL aliquot was injected onto the precolumn. Double distilled water was used to elute and remove proteins and polar components in the sample. The components retained on the precolumn were flushed forward onto the analytical column by the mobile phase (acetonitrile-0.2 mol/L ammonium sulphate, 10:90 v/v) with automated column switching. The limit of determination of pseudoephedrine in plasma was 12 ng/mL. The relative standard deviations of intra- and inter-assay for the determination of pseudoephedrine in plasma were 1.2-9.8% over the concentration range 1020-21.8 ng/mL. The mean recovery by on-line solid phase extraction was 94.76% (RSD = 1.1%).     

Alcohols and wide-bore capillaries in nonaqueous capillary electrophoresis.

The feasibility of using C1-C5 alcohols as electrolyte solutions in nonaqueous capillary zone electrophoresis was investigated. The separation of basic narcotic analgesics and acidic diuretics was modified by changing the alcohol in an electrolyte solution containing alcohol-acetonitrile-acetic acid (50:49:1, v/v) and 20 mM ammonium acetate while other experimental conditions were kept constant. The alcohols studied were methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, and 1-pentanol. The results indicate that even longer-chain alcohols can be used in nonaqueous capillary zone electrophoresis and, because of the lower currents they allow, they are especially advantageous in wider capillaries. Basic analytes were separated in 200 microm and 320 microm ID capillaries with 1-butanol-acetonitrile-acetic acid (50:49:1, v/v) containing 20 mM ammonium acetate as electrolyte solution. Problems related to the use of wide-bore capillaries are discussed.     

Simultaneous determination of diethylene glycol and propylene glycol in pharmaceutical products by HPLC after precolumn derivatization with p-toluenesulfonyl isocyanate

A simple and reliable HPLC method was developed for the simultaneous quantitative analysis of diethylene glycol (DEG) and propylene glycol (PG) in pharmaceutical products by precolumn derivatization. The derivatization reagent p-toluenesulfonyl isocyanate (TSIC, 10 microL, 20% in ACN v/v) was added to 100 microL of the sample, and then 10 muL of water was added. The resulting derivatives were separated using a C(18)analytical column and a mobile phase composed of 0.01 M KH(2)PO(4)buffer (adjusted to pH 2.5 with phosphoric acid) and ACN (47:53 v/v) at 1 mL/min and 25 degrees C. For detection, UV light at 227 nm was used. The derivatization conditions including reaction time, temperature, and concentration of TSIC were optimized. The calibration curves were linear from 0.062 to 18.6 microg/mL (r(2)= 0.9999) and from 0.071 to 21.3 microg/mL (r(2) = 0.9999) for DEG and PG, respectively. The RSD values of intra- and interday assays were all below 4% for DEG and PG. The proposed method was then successfully applied to analyze two Armillarisin A injection samples and two spiked syrup samples.     

高效液相色谱法测定血清中头孢噻肟浓度

用固相萃取法处理样品,以扑热息痛为内标物、甲醇－醋酸钠／醋酸缓冲溶液作流动相,采用反相高效液相色谱法测定血清中头孢噻肟的浓度。头孢噻肟和内标物的平均回收率分别为９６．７％和９７．７％,头孢噻肟血清浓度在１０ｍｇ／Ｌ至１５０ｍｇ／Ｌ范围内有良好线性关系,最低检测浓度为２ｍｇ／Ｌ,日内和日间相对标准偏差分别在３．０％和４．１％以内。结果表明方法准确、简便。     

Determination of monophosphate nucleotides by capillary electrophoresis inductively coupled plasma mass spectrometry

A preliminary study of a modified microconcentric nebulizer (CEI-100, CETAC) as the sample introduction device of capillary electrophoresis inductively coupled plasma mass spectrometry (CE-ICP-MS) for the determination of monophosphate nucleotides is described. The monophosphate nucleotides studied include adenosine 5'-monophosphate (AMP), guanosine 5'-monophosphate (GMP), uridine 5'-monophosphate (UMP) and inosine 5'-monophosphate (IMP). The species studied were well separated using a 70 cm length x 75 microm id fused silica capillary while the applied voltage was set at -22 kV and a 20 mmol l(-1) ammonium citrate/citric acid buffer (pH 4.0) containing 0.1% m/v cationic polymer (hexadimethrine bromide, Polybrene) was used as the electrophoretic buffer. The electroosmotic flow was reversed by flushing the fused silica capillary with 0.2% m/v Polybrene to accelerate separation. The detection limit of various species studied was in the range of 0.036-0.054 microg P ml(-1), which corresponded to the absolute detection limit of 1.1-1.6 pg P based on the injection volume of 30 nl. We determined the concentrations of nucleotides in two IG-enriched monosodium glutamates purchased from the local market. The recovery was in the range of 100-112% for various species, and the concentrations of IMP and GMP in these samples were in the range of 0.15-0.18% m/m.