Capillary high-performance liquid chromatography/electrospray ion trap time-of-flight mass spectrometry using a novel nanoflow gradient generator

A type of high-performance liquid chromatography (HPLC) based on a novel nanoflow gradient generator (Asymptotic-Trace-10-Port-Valve (AT10PV) nanoGR generator) was developed and coupled with an electrospray ion trap time-of-flight mass spectrometer (ESI-IT-TOF MS). Stability of the nanoflow GR HPLC system was tested at flow rates of 20 and 50 nL/min by using a nanoflow meter. Average flow rates in a 2-h run were 51.2 nL/min with RSD 0.7% and 21.0 nL/min with RSD 1.8%. Repeatability of analysis of the nanoHPLC/ESI-IT-TOF MS system was also tested by injecting 1.0 microL of trypsin digested bovine serum albumin (BSA) (100 fmol) into a monolithic silica-ODS column (30 microm i.d., 150 mm in length) through a packed silica-ODS trapping column (particle size 5 microm, 150 microm i.d., 10 mm in length). At a flow rate of 50 nL/min, the result demonstrated a reasonably good repeatability of peak retention times (RSD: 0.32-1.1%) and base-ion peak areas (RSD: 4.4-6.6%).     

On-line capillary separations/tandem mass spectrometry for protein digest analysis by using an ion trap storage/reflectron time-of flight mass detector

Capillary separations interfaced to tandem mass spectrometry provide a very powerful tool for the characterization of biological macromolecules such as proteins and peptides. The development of real time data-dependent data acquisition has further enhanced the capability of this method. However, the application of this technique to fast capillary separations has been limited by the relatively slow spectral acquisition speed available on scanning mass spectrometers. In this work, an ion trap storage/reflectron time-of-flight mass spectrometer (IT/reTOF-MS) has been used as an on-line tandem mass detector for capillary high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) separations of peptide mixtures including a protein digest. By taking advantage of the nonscanning property of the time-of-flight mass spectrometer, a fast spectral acquisition rate has been achieved. This fast spectral acquisition rate, combined with a new protocol that speeds up tickle voltage optimization, has provided MS/MS spectra for multiple components in a hemoglobin digest during one liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) run. Further, the IT/reTOF-MS has the speed to provide MS/MS spectra for multiple components in a CE separation of a synthetic peptide mixture within one CE/MS/MS run.     

High-throughput determination of atrasentan in human plasma by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

Atrasentan (A-147627) is an endothelin antagonist receptor being developed at Abbott Laboratories for the treatment of prostate cancer. A quick and sensitive method for the determination of atrasentan in human plasma has been developed and validated using high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry. A dual-column, single mass spectrometer system is used to provide a reliable and routine means to increase sample throughput. The analytical method involves liquid-liquid extraction and internal standard (A-166790). The plasma samples and internal standard are acidified with 0.3 M hydrochloric acid prior to being extracted into 1:1 (v/v) hexanes--methyl t-butyl ether. The organic extract was evaporated to dryness using heated nitrogen stream and reconstituted with mobile phase. Atrasentan and internal standard were separated with no interference in a Zorbax SB-C(18) analytical column with 2.1 x 50 mm, 5 microm, and a Zorbax C(8) guard column using a mobile phase consisting of 50:50 (v:v) acetonitrile--0.05 M ammonium acetate, pH 4.5, at a flow rate of 0.30 mL/min to provide 4 min chromatograms. For a 250 microL plasma sample volume, the limit of quantitation was approximately 0.3 ng/mL. The calibration was linear from 0.30 to 98.0 ng/mL (r(2) > 0.995). A significant advantage of the method is the ability to employ parallel HPLC separations with detection by a single MS/MS system to provide sensitivity and selectivity sufficient to achieve robust analytical results with a lower limit of quantitation of 0.30 ng/mL and high throughput.     

The rapid identification of drug metabolites using capillary liquid chromatography coupled to an ion trap mass spectrometer

Capillary liquid chromatography (LC) using a 320 microns column and a flow rate of 10 microL/min has been coupled to an ion trap mass spectrometer using electrospray ionisation (ESI) to enable the rapid and effective identification of metabolites in urine, following oral administration of a novel human neutrophil elastase inhibitor, GW311616. Metabolites were identified from their mass (MS) spectra and tandem (MS/MS) mass spectra using minimal sample (1 microL of urine) and no sample pretreatment. Sensitivity assessment has shown that both molecular weight and structural information is obtainable on as little as 5 pg of compound, making the capillary LC/ion trap system as described an ideal analytical tool for the detection and characterisation of low level metabolites in biofluids (particularly when sample volume is limited). This level of detection was unattainable using a triple quadrupole mass spectrometer operating in full-scan mode, although 200 fg on column was detected using selected reaction monitoring target analysis.     

A novel interface for variable flow nanoscale LC/MS/MS for improved proteome coverage

A variable flow "peak trapping" liquid chromatography (LC) interface has been developed for the coupling of nanoscale LC to electrospray ionization mass spectrometry (ESI-MS). The presented peak trapping LC interface allows for the extended analysis time of co-eluting compounds and has been employed for the identification of proteins via tandem mass spectrometry (MS/MS). The variable flow process can be controlled either manually or in a completely automated manner where the mass spectrometer status determines the status of the variable flow interface. When the mass spectrometer operates in MS survey mode, the interface is operated in a so-called "high-flow" mode. Alternatively, the interface is operated in a "low-flow" mode during MS/MS analysis. In the "high-flow" mode of the variable flow process the column flow rate is typically around 200 nL/min, whereas in the "low-flow" mode the column effluent is introduced into the source of the mass spectrometer at 25 nL/min. In addition to the flow reduction during MS/MS analysis, the gradient is paused to preserve the peptide separation on the analytical nanoscale LC column. The performance of the variable flow nanoscale LC/MS/MS interface is demonstrated by the automated analysis of standard peptide mixtures and protein digests utilizing variable flow, data-dependent scanning MS/MS techniques, and automated database searching.     

An exponential dilution gradient system for nanoscale liquid chromatography in combination with MALDI or Nano-ESI mass spectrometry for proteolytic digests

A simple and inexpensive nano high performance liquid chromatography system (nano-LC) employing the exponential dilution method for gradient separations was built. The system was used to analyze a tryptic digest of Escherichia coli uracil DNA glycosylase (Ung; Mr = 25,563), a DNA-binding protein that initiates the uracil-excision DNA repair process by catalyzing the release of uracil from the deoxyribose phosphate backbone of DNA. Both on-line and off-line approaches to analyzing peptides produced by in-gel digestion of Ung are demonstrated. The on-line approach uses nano-high performance liquid chromatography (HPLC)/micro-electrospray MS to assign peptide masses. The off-line approach uses matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and nano-electrospray/collision-induced dissociation (CID) tandem mass spectrometry, to analyze fractions (2-3 microL) collected manually from the nano-LC system. The nano-electrospray technique allows detailed fragmentation information to be obtained at different collision energies with only a marginal increase in sample handling due to the nano-LC step.     

Separation and identification of 20 chemical constituents in the traditional Chinese medicinal preparation Shenbao tablet by LC-ESI-MS3

A sensitive and specific high-performance liquid chromatography (HPLC)-electrospray ionization multiple-stage mass spectrometry for the simultaneous separation and identification of 20 chemical constituents in the traditional Chinese medicinal preparation of the Shenbao tablet is established. The samples are separated with an Alltima C(18) column (250 x 4.6 mm, 5 microm) by linear gradient elution using water-acetic acid (A; 100:0.5, v/v) and acetonitrile (B; 0 min, 76:24; 15 min, 70:30; 40 min, 53:47; 50 min, 30:70; and maintain 10 min) as the mobile phase at a flow rate of 1.0 mL/min. The ion trap mass spectrometer is coupled to the HPLC system. Satisfactory results are obtained within 60 min for the simultaneous separation and identification of the 20 constituents. This is the first report on the analysis of main chemical constituents in the Shenbao tablet.     

Evaluation of several solid phase extraction liquid chromatography/tandem mass spectrometry on-line configurations for high-throughput analysis of acidic and basic drugs in rat plasma

Several configurations using 6- and 10-port switching valves were studied for high flow, on-line extraction of rat plasma coupled to an electrospray triple quadrupole mass spectrometer. Each plasma sample was diluted 1:1 with an aqueous internal standard solution. The sample was injected into a 2.1 x 20 mm cartridge column packed with 25 microm divinylbenzene/N-vinylpyrrolidone packing using 100% aqueous mobile phase at 4 mL/min. After sample loading and sample cleanup, the analytes were eluted from the extraction column with a 1.0-min gradient at 0.4 mL/min. The samples were either analyzed directly after elution from the extraction column or after additional separation using a short high performance liquid chromatography (HPLC) column. The different configurations were tested using an acidic drug (diflunisal) and a basic drug (clemastine) in rat plasma. On-line analysis was performed by injecting 200 microL of diluted plasma. The mass spectrometer was operated in the multiple reaction monitoring (MRM) mode. All calibration standards gave relative standard deviations (RSDs) below 5%. The total time per sample was 3 min.     

A simple and sensitive assay for the quantitative analysis of rivastigmine and its metabolite NAP 226-90 in human EDTA plasma using coupled liquid chromatography and tandem mass spectrometry

A sensitive and specific high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) assay for the determination of rivastigmine and its major metabolite NAP 226-90 is presented. A 100 microL plasma aliquot was spiked with a structural analogue of rivastigmine as internal standard (PKF214-976-AE-1) and proteins were precipitated by adding 200 microL of methanol. After centrifugation a volume of 100 microL of the clear supernatant was mixed with 100 microL of methanol/water (30:70, v/v) and volumes of 25 microL were injected onto the HPLC system. Separation was acquired on a 150 x 2.0 mm i.d. Gemini C18 column using a gradient system with 10 mM ammonium hydroxide and methanol. Detection was performed by using a turboionspray interface and positive ion multiple reaction monitoring by tandem mass spectrometry. The assay quantifies rivastigmine from 0.25 to 50 ng/mL and its metabolite NAP 226-90 from 0.50 to 25 ng/mL, using human plasma samples of 100 microL. Validation results demonstrate that rivastigmine and metabolite concentrations can be accurately and precisely quantified in human EDTA plasma. This assay is now used to support clinical pharmacologic studies with rivastigmine.     

高效液相色谱-质谱法分析菊芋叶中的绿原酸类化合物

建立了菊芋叶中绿原酸类化合物的高效液相色谱-紫外检测-质谱(HPLC-UV-MS)定性分析方法。液相色谱条件:Inertsil ODS-3色谱柱(250 mm×4.6 mm,5 μm);甲醇和水(含1%乙酸)梯度洗脱,流量1.0 mL/min;柱温35 ℃;检测波长327 nm。质谱条件:Thermo公司TSQ三级四极杆质谱仪;电喷雾电离(ESI)接口;负离子检出模式。采用该方法得到了菊芋叶提取物的紫外检测的色谱图、负离子监测的总离子流图以及相应色谱峰的紫外光谱图和一级、二级质谱图,对其进行解析,鉴别出菊芋叶中的7个绿原酸类成分。该方法简便、快速、灵敏度高,可以很好地对菊芋叶中的绿原酸类化合物进行定性分析。     

On-line extraction coupled with liquid chromatography tandem mass spectrometry for quantitation of pravastatin and its metabolite in human serum

A high-throughput bioanalytical method for simultaneous quantitation of pravastatin and its metabolite (M1) in human serum was developed and validated using on-line extraction following liquid chromatography tandem mass spectrometry (LC-MS/MS). The on-line extraction was accomplished by the direct injection of a 50 microL serum sample, mixed 4:1 with an aqueous internal standard solution, into one of the extraction columns with aqueous 1 mm formic acid at flow rate of 3 mL/min. The separation and analysis were achieved by back-eluting the analytes from the extraction column and the analytical column to the mass spectrometer with an isocratic mobile phase consisting of 62% aqueous 1 mm formic acid and 38% acetonitrile at a flow rate of 0.8 mL/min. The second extraction column was being equilibrated while the first column was being used for analysis, and vice versa. The standard curve range was 0.500-100 ng/mL for pravastatin and M1. The lower limit of quantitation, 0.500 ng/mL for all the analytes, was achieved when 50 microL of human serum was used. The intra- and inter-day precisions were within 7.4%, and the accuracy was between 95 and 103%. The on-line extraction was finished in 0.5 min and total analysis time was 2.5 min per sample.     

Increasing throughput of parallel on-line extraction liquid chromatography/electrospray ionization tandem mass spectrometry system for GLP quantitative bioanalysis in drug development

An approach is described with turbulent flow on-line extraction liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) for GLP quantitative bioanalysis of a drug candidate. Two systems were built in-house with standard laboratory parts and equipments. One system consisted of one gradient HPLC pump, one isocratic pump, one ten-port valve, two turbulent flow columns, one analytical column, one autosampler and one mass spectrometer. Using this system, an injection-to-injection cycle time of 0.8 min was achieved. By adding an additional valve, another analytical column and an isocratic pump, the injection-to-injection cycle time decreased to 0.4 min. Validation results from the two systems showed that precision and accuracy were acceptable for GLP quantitative analyses. The system was utilized to support sample bioanalysis of a drug candidate in a first-time in-human clinical trial.     

Capillary electrophoresis-atmospheric pressure ionization mass spectrometry for the characterization of peptides. Instrumental considerations for mass spectrometric detection.

On-line capillary electrophoresis (CE) separations are shown for a synthetic peptide mixture and a tryptic digest of human hemoglobin in an uncoated fused-silica capillary with detection using atmospheric pressure ionization mass spectrometry (API-MS). The CE system utilized a 1-m capillary column of either 75- or 100-microns I.D. These somewhat larger inside diameters allow higher sample capacities for MS detection and the 1-m length facilitates connecting the CE column to the liquid junction-ion spray interface and MS system. Low volatile buffer concentrations (15-20 mM) of ammonium acetate or ammonium formate, and high organic modifier content (5-50%) of methanol or acetonitrile facilitates ionization under electrospray conditions. This study shows that peptides separated by CE may be transferred to the API-MS system through a liquid junction coupling to the pneumatically assisted electrospray (ion spray) interface at low buffer pH when the electroosmotic flow is low (0-0.04 microliter/min). CE-MS as described herein is facilitated by features in modern CE instrumentation including robotic cleaning and pressurization of the capillary inlet. The latter is particularly useful for repetitive rinsing and conditioning of the capillary column between analyses in addition to continuous 'infusion' of sample to the mass spectrometer for tuning purposes. In addition to facile molecular weight determination, amino acid sequence information for peptides may be obtained by utilizing on-line tandem MS. After the tryptic digest sample components enter the API-MS system, the molecular ion species of individual peptides may be focussed and transmitted into the collision cell of the tandem triple quadrupole mass spectrometer. Collision-induced dissociation of protonated peptide molecules yielded structural information for their characterization following injection of 10 pmol of a tryptic digest from human hemoglobin.     

Effects of flow rate on high-throughput quantitative analysis of protein-precipitated plasma using liquid chromatography/tandem mass spectrometry

The effects of flow rate and column length on analyte response (peak area and height), total cycle time, column backpressure, and elution volume are presented. Rapid chromatographic separations and tandem mass spectrometric (MS/MS) detection are applied to the supernatant of protein-precipitated plasma standards containing four compounds from a drug discovery screen. The plasma samples were injected onto three C-18 columns (2 x 10,2.1 x 30 and 2.1 x 50 mm) at flow rates of 0.25, 0.50, 1.00 and 1.50 mL/min. The plasma samples were detected using a Sciex API 3000 tandem mass spectrometer operated in the Turbo Ionspray mode. A post-column split was used to maintain a flow rate of 0.25 mL/min into the mass spectrometer source to avoid differences in nebulization efficiency. The data show that diluted protein-precipitated plasma supernatants show average matrix effects (i.e. suppression) of 60.0% (2 x 10 mm), 89.3% (2 x 30 mm), and 76.7% (2 x 50 mm) of expected response at 10 ng/mL. Average matrix effects of 70.2% (2 x 10 mm), 88.9% (2 x 30 mm), and 81.2% (2 x 50 mm) of expected response at 1000 ng/mL plasma. The data also show if peak widths remain relatively constant, analytes are less sensitive as flow rates are increased. These data are consistent with the concentration-dependent relationship of ionspray in the range of flow rates studied. The data show that, while analyte response decreased proportionately to increases in flow rate, the analysis cycle times did not decrease proportionately.     

Simultaneous determination of cucurbitacin IIa and cucurbitacin IIb of Hemsleya amabilis by HPLC–MS/MS and their pharmacokinetic study in normal and indomethacin‐induced rats

A selective and sensitive HPLC–MS/MS method was developed for the simultaneous determination of cucurbitacin IIa (cuIIa) and cucurbitacin IIb (cuIIb), the major bioactive cucurbitacins of Hemsleya amabilis, in rat plasma using euphadienol as internal standard (IS). After liquid–liquid extraction with dichloromethane, separation was achieved on a Syncronis HPLC C₁₈ column (150 mm × 4.6 mm, 5 μm) using an isocratic mobile phase system consisting of acetonitrile–water (85:15, v/v) at a flow rate of 0.6 mL/min with a split ratio of 1:2. Detection was performed on a TSQ Quantum Ultra mass spectrometer equipped with an positive‐ion electrospray ionization source. The lower limits of quantification (LLOQs) were 0.25 and 0.15 ng/mL for cuIIa and cuIIb, respectively. The intra‐ and inter‐day precision was <11.5% for the LLOQs and each quality control level of the analytes, and accuracy was between ?9.1 and 7.6%. The extraction recoveries of the analytes and IS from rat plasma were all >87.1%. The method was fully validated and applied to compare the pharmacokinetic profiles of the two cucurbitacins in rat plasma after oral administration of H. amabilis extract between normal and indomethacin‐induced rats. Copyright © 2016 John Wiley & Sons, Ltd.     

Optimizing the peak capacity per unit time in one-dimensional and off-line two-dimensional liquid chromatography for the separation of complex peptide samples

To obtain the best compromise between peak capacity and analysis time in one-dimensional and two-dimensional (2D) liquid chromatography (LC), column technology and operating conditions were optimized. The effects of gradient time, flow rate, column temperature, and column length were investigated in one-dimensional reversed-phase (RP) gradient nano-LC, with the aim of maximizing the peak per unit time for peptide separations. An off-line two-dimensional LC approach was developed using a micro-fractionation option of the autosampler, which allowed automatic fractionation of peptides after a first-dimension ion-exchange separation and re-injection of the fractions onto a second-dimension RP nano-LC column. Under the applied conditions, which included a preconcentration/desalting time of 5 min, and a column equilibration time of 12.5 min, the highest peak capacity per unit time in the 2D-LC mode was obtained when applying a short (10 min) first-dimension gradient and second-dimension RP gradients of 20 min duration. For separations requiring a maximum peak capacity of 375, one-dimensional LC was found to be superior to the off-line strong cation-exchange/×/RPLC approach in terms of analysis time. Although a peak capacity of 450 could be obtained in one-dimensional LC when applying 120-min gradients on 500-mm long columns packed with 3-μm particles, for separations requiring a peak capacity higher than 375 2D-LC experiments provide a higher peak capacity per unit time. Finally, the potential of off-line 2D-LC coupled to tandem mass spectrometry detection is demonstrated with the analysis of a tryptic digest of a mixture of nine proteins and an Escherichia coli digest.     

Simultaneous quantification of fludarabine and cyclophosphamide in human plasma by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

Fludarabine and cyclophosphamide are anticancer agents mainly used in the treatment of hematologic malignancies. We have developed and validated an assay using high-performance liquid chromatography (HPLC) coupled with electrospray ionization tandem mass spectrometry for the quantification of fludarabine in combination with cyclophosphamide in human heparin and human EDTA plasma. Sample pre-treatment consisted of a protein precipitation with cold acetonitrile (-20 degrees C) using 250 microL of plasma. Separation was performed on an Extend C18 column (150 x 2.1 mm i.d.; 5 microm) with a stepwise gradient using 1 mM ammonia solution and acetonitrile at a flow rate of 400 microL/min. The analytical run time was 12 min. The triple quadrupole mass spectrometer was operated in the positive ion mode and multiple reaction monitoring was used for drug quantification. The method was validated over a concentration range of 1 to 100 ng/mL for fludarabine and cyclophosphamide in human heparin and human EDTA plasma. The coefficients of variation were <13.9% for inter- and intra-day precisions. Mean accuracies were also within the designated limits (+/-15%). The analytes were stable in plasma, processed extracts and in stock solution under all relevant conditions.     

Fast, generic gradient high performance liquid chromatography coupled to fourier transform ion cyclotron resonance mass spectrometry for the accurate mass analysis of mixtures

Fast gradient high performance liquid chromatography (HPLC) has been combined with a commercially available Fourier transform ion cyclotron resonance (FTICR) mass spectrometer for the routine and high performance analysis of mixtures. With this combination we were able to separate and detect, under high mass accuracy conditions, a six-component drug mixture in less than 5 minutes. The fast gradients described are now possible due to the development of mechanically robust, ultra pure silica packing materials, which allow relatively high flow rates (ca. 1 mL/min for a 2 mm diameter column). For the six compounds present in the model mixture, relative mass errors of less than 1 ppm were obtained (based on an external calibration) providing sufficient mass accuracy to make unequivocal assignments of empirical formulae. Preliminary results of fast gradient HPLC/FTICR-MS/MS are also shown for the same six-component mixture. Copyright 2000 John Wiley & Sons, Ltd.     

Fully automated analysis of estrogens in environmental waters by in-tube solid-phase microextraction coupled with liquid chromatography-tandem mass spectrometry

A simple, rapid and sensitive method for the determination of five estrogens, estrone, 17beta-estradiol, estriol, ethynyl estradiol, and diethylstilbestrol, was developed using a fully automated method consisting of in-tube solid-phase microextraction (SPME) coupled with liquid chromatography-tandem mass spectrometry (LC/MS/MS). These estrogens were separated within 8 min by HPLC using an XDB-C8 column and 0.01% ammonia/acetonitrile (60/40, v/v) at a flow rate of 0.2 mL/min. Electrospray ionization conditions in the negative ion mode were optimized for MS/MS detection of the estrogens. The optimum in-tube SPME conditions were 20 draw/eject cycles of 40 microL of sample using a Supel-Q PLOT capillary column as an extraction device. The extracted compounds were easily desorbed from the capillary by passage of the mobile phase, and no carryover was observed. Using the in-tube SPME LC/MS/MS method, good linearity of the calibration curve (r > or = 0.9996) was obtained in the concentration range from 10 to 200 pg/mL for all compounds examined. The limits of detection (S/N= 3) of the five estrogens examined ranged from 2.7 to 11.7 pg/mL. The in-tube SPME method showed 34-90-fold higher sensitivity than the direct injection method (5 microL injection). This method was applied successfully to the analysis of environmental water samples without any other pretreatment and interference peaks. Several surface water and wastewater samples were collected from the area around Asahi River, and estriol was detected at 35.7 pg/mL in the effluent of a sewage treatment plant. The recoveries of estrogens spiked into river waters were above 86%, except for estriol, and the relative standard deviations were below 0.9-8.8%.     

Rapid high-performance liquid chromatography-electrospray ionization tandem mass spectrometry method for qualitative and quantitative analysis of virgin olive oil phenolic metabolites in human low-density lipoproteins

A rapid method for detection and quantification of metabolites of specific olive oil phenolic compounds (hydroxytyrosol monoglucuronide, hydroxytyrosol monosulfate, tyrosol glucuronide, tyrosol sulfate and homovanillic acid sulfate) in low-density lipoprotein (LDL) fractions by solid-phase extraction (SPE) and high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS) is described. A 3 microm particle size fast C18 Luna column, 5 cm x 2.0 mm I.D., was used at a flow rate of 0.6 mL/min with a mobile phase consisting of 0.1% (v/v) formic acid (A) and acetonitrile (B). A linear gradient profile was used for separation at column temperature 40 degrees C. The proposed chromatographic procedure is rapid without loosing its separation efficiency and sensitivity. Validation proofs were carried out for the method described, showing a linear system (r>0.99) and a recovery of 81.9 and 101.3% for hydroxytyrosol and homovanillic acid, respectively. The results show that this method is effective and can be used in routine analysis.