Quality evaluation of Guan‐Xin‐Ning injection based on fingerprint analysis and simultaneous separation and determination of seven bioactive constituents by capillary electrophoresis

The purpose of this study was to develop a comprehensive, rapid and practical capillary electrophoresis (CE) method for quality control (QC) of Guan‐Xin‐Ning (GXN) injection based on fingerprint analysis and simultaneous separation and determination of seven constituents. In fingerprint analysis, a capillary zone electrophoresis (CZE) method with a running buffer of 30 mM borate solution (pH 9.3) was established. Meanwhile, ten batches of samples were used to establish the fingerprint electropherogram and 34 common peaks were obtained within 20 min. The RSD of relative migration times (RMT) and relative peak areas (RPA) were less than 5%. In order to further evaluate the quality of GXN injection, a micellar electrokinetic chromatography (MEKC) method was developed for simultaneous separation and determination of bioactive constituents. Seven components reached baseline separation with a running buffer containing 35 mM SDS and 45 mM borate solution (pH 9.3). A good linearity was obtained with correlation coefficients from 0.9906 to 0.9997. The LOD and LOQ ranged from 0.12 to 1.50 μg/mL and from 0.40 to 4.90 μg/mL, respectively. The recoveries ranged between 99.0 and 104.4%. Therefore, it was concluded that the proposed method can be used for full‐scale quality analysis of GXN injection.     

Non-aqueous capillary electrophoresis for separation and simultaneous determination of fraxin, esculin and esculetin in Cortex fraxini and its medicinal preparations

A non-aqueous capillary electrophoresis method has been developed for the separation and simultaneous determination of fraxin, esculin and esculetin in Cortex fraxini and its preparation for the first time. Optimum separation of the analytes was obtained on a 47 cm x 75 microm i.d. fused-silica capillary using a non-aqueous buffer system of 60 mM sodium cholate, 20 mM ammonium acetate, 20% acetonitrile and 3% acetic acid at 20 kV and 292 K, respectively. The relative standard deviations (RSDs) of the migration times and the peak heights of the three analytes were in the range of 0.23-0.28 and 2.12-2.60%, respectively. Detection limits of fraxin, esculin and esculetin were 0.1557, 0.4073 and 0.5382 microg/mL, respectively. In the tested concentration range, good linear relationships (correlation coefficients 0.9995 for fraxin, 0.9999 for esculin and 0.9992 for esculetin) between peak heights and concentrations of the analytes were observed. This method has been successfully applied to simultaneous determination of the three bioactive components with the recoveries from 90.2 to 109.2% in the five samples.     

Simultaneous determination of seven phenolic acids in three Salvia species by capillary zone electrophoresis with β‐cyclodextrin as modifier

A capillary zone electrophoresis method was developed for the simultaneous determination of seven phenolic acids, including protocatechuic aldehyde ( 1 ), salvianolic acid C ( 2 ), rosmarinic acid ( 3 ), salvianolic acid A ( 4 ), danshensu ( 5 ), salvianolic acid B ( 6 ), and protocatechuic acid ( 7 ), in Danshen and related medicinal plants. A running buffer composed of 20 mM sodium tetraborate adjusted to pH 9.0, and containing 12 mM β‐cyclodextrin as modifier. Baseline separation was achieved within 17 min running at the voltage of 20 kV, temperature of 25°C and detection wavelength of 280 nm. The relative standard deviations of migration time ranged from 0.2 to 0.7% and the peak area ranged from 1.5 to 3.7% for the seven analytes, indicating the good repeatability of the proposed method. The method was extensively validated by evaluating the linearity (R² ≥ 0.9992), limits of detection (0.14–0.36 μg/mL), limits of quantification (0.47–1.19 μg/mL), and recovery (96.0–102.6%). Under the optimum conditions, samples of Danshen and related medicinal plants were analyzed using the developed method with high separation efficiency.     

Determination of six bioactive components of Saussurea katochaete by capillary electrophoresis

The simultaneous determination of 3,4-dimethoxy-3'-hydroxy propiophenone, 5-hydroxy-7-methoxy coumarin, 7-hydroxy coumarin, 3',5'-dimethoxy apigenin, apigenin and 4-hydroxy cinnamic acid in the extract of S. katochaete has been investigated by capillary electrophoresis for the first time. The six active components were completely separated within 10 min in 20 mM Na(2)HPO(4) buffer at pH 11.00 with 10% (v/v) methanol and detected at 214 nm. The applied voltage was 15 kV and the temperature was kept at 25 degrees C. The effects of buffer pH, the concentration of Na(2)HPO(4) and the concentration of methanol on the separation efficiency were studied systematically. The regression equations revealed good linear relationships (correlation coefficients were 0.9987-0.9998) between the peak area of each analyte and its concentration. The relative standard deviations (RSD) of migration time and peak areas were <1.63 and <4.03%, respectively. The application of this method for the separation and determination of the six bioactive components in S. katochaete was reported. The contents of the six analytes ranged from 0.023 to 0.131 mg/g and recoveries ranged from 94.7 to 104.8%.     

Enhanced analysis of triterpenes, flavonoids and phenolic compounds in Prunella vulgaris L. by capillary zone electrophoresis with the addition of running buffer modifiers

A cyclodextrin-modified capillary zone electrophoresis method was developed for the separation and determination of three isomeric compounds (ursolic acid, oleanolic acid and betulinic acid), caffeic acid, p-coumaric acid, rosmarinic acid, rutin and quercetin. Without the addition of beta-cyclodextrin (beta-CD) and methanol, the separation of these analytes was poorly resolved. These eight compounds, however, were well separated from each other within 20 min with a borax running buffer (40 mM of borax, pH 9.4) containing 2mM beta-CD and 4% (v/v) methanol at the voltage of 25 kV, temperature of 25 degrees C and detection wavelength of 210 nm. The relative standard deviations (RSDs) of migration time ranged from 0.16 to 0.74% while those of the peak area ratios ranged from 2.17 to 4.61% for six determinations of the analytes at concentration of 10 and 25 microg mL(-1). The correlation coefficients of the calibration curves of the analytes were all >0.998, and the recoveries were from 96.8 to 103.6%. The method was successfully applied to determine these bioactive components in the samples of Prunella vulgaris L. and its beverage drink products. Our results reveal that only the isomeric compounds and rosmarinic acid could be detected in the spikes of P. vulgaris L.; other components were either too low to be detected or not present while only rosmarinic acid was detected in the beverage products.     

Capacitively coupled contactless conductivity detection as an alternative detection mode in CE for the analysis of kanamycin sulphate and its related substances

A method was developed to determine simultaneously kanamycin, its related substances and sulphate in kanamycin sulphate using capacitively coupled contactless conductivity detection. Kanamycin is an aminoglycoside antibiotic that lacks a strong UV-absorbing chromophore. Due to its physicochemical properties, CE in combination with capacitively coupled contactless conductivity detection was chosen. The separation method uses a BGE composed of 40 mM 2-(N-morpholino)ethanesulphonic acid monohydrate and 40 mM L-histidine, pH 6.35. A 0.6 mM N-cetyltrimethyl ammonium bromide (CTAB) solution was added as electroosmotic flow modifier in a concentration below the critical micellar concentration (CMC). Ammonium acetate 50 mg/L was used as internal standard. In total, 30 kV was applied in reverse polarity on a fused-silica capillary (65/41 cm; 75 μm id). The optimized separation was obtained in less than 6 min with good linearity (R(2)=0.9999) for kanamycin. It shows a good precision expressed as RSD on the relative peak areas equal to 0.3 and 1.1% for intra-day and inter-day precision, respectively. The LOD and LOQ are 0.7 and 2.3 mg/L, respectively. Similarly, for sulphate, a good linearity (R(2)=0.9996) and precision (RSD 0.4 and 0.6% for intra-day and inter-day, respectively) were obtained.     

Determination of homologues of quaternary ammonium surfactants by capillary electrophoresis using indirect UV detection

This investigation describes the simultaneous separation of two major non-chromophoric quaternary ammonium surfactants, alkyltrimethyl- and dialkyldimethylammonium compounds (ATMACs and DADMACs, respectively), by capillary electrophoresis (CE) using indirect UV detection. The most effective separation conditions was 10 mM phosphate buffer with 57.5% tetrahydrofuran and 3 mM sodium dodecyl sulfate (SDS) at pH 4.3, and the sample hydrodynamic injection of up to 20 s at 1 psi (approximately 60 nl), and an applied voltage of 25 kV (1 psi = 6.9 kPa). Specially, the selection of an appropriate chromophore and an internal standard (I.S.) to improve the peak identification and quantitation was systematically investigated. Decylbenzyldimethyl ammonium chloride (C10-BDMA+C-) as a chromophore with 3 mM sodium dodecyl sulfate provided the best detectability for all homologues. The reproducibility of the migration time and quantitative analysis can be improved by using tetraoctyl ammonium ion as an internal standard, giving the relative standard deviation (R.S.D.) less than 0.8% for the relative migration times, and 2.5-5.5% for the relative peak areas. A good linearity of CE analysis was obtained in the range of 1.0-20 microg/ml with r2 values of above 0.999. The analysis of cationic surfactants in commercial products of hair conditioners and fabric softeners was also performed. Electrospray mass spectrometric method was applied to evaluate the CE method, and the compatible results were obtained.     

气相色谱-三重四极杆质谱法同时测定烟丝中73种香气物质

建立了气相色谱-三重四极杆质谱(GC-MS/MS)同时测定烟丝中73种香气物质的分析方法。样品用无水乙醚振荡提取,提取液经过滤后直接进入DB-5MS色谱柱(30 m×0.25 mm,0.25μm)分离,通过优化质谱参数,可有效降低复杂基质和重叠峰的干扰,同时采用多反应监测(MRM)模式测定,内标法定量。结果表明,目标物在各自的线性范围内线性关系良好(r~20.99),低、中、高3个水平的加标回收率为70.0%~122.3%,相对标准偏差(RSD)为1.6%~20.4%。该方法具有前处理简单、准确灵敏的特点,适用于烟丝样品中73种香气物质的检测。     

Determination of Dexamethasone in Cosmetics by MEKC

A rapid and reliable method based on micellar electrokinetic capillary chromatography has been developed for the determination of dexamethasone in cosmetics. Effects of buffer composition, concentration and pH, the detection wavelength, separation voltage, and injection time were systematically investigated. The optimum conditions were: 30 mM borax buffer containing 20 mM sodium dodecyl sulfate at pH 9.0, detection at 254 nm, injection time 10 s at a height of 10 cm, and a separation voltage of 15 kV. Under these conditions, the analysis of dexamethasone in cosmetics was carried out within 6 min. The method was validated for stability, precision, linearity and accuracy. Excellent linearity was obtained in the range of 50–1,000 μg mL⁻¹, and acceptable precision, in intra-day and inter-day analysis, was also obtained with relative standard deviation in the range of 0.19–0.86 and 2.50–4.90% for migration time and peak area ratio, respectively. The method was used to analyse eight cosmetic samples purchased locally.     

Simultaneous determination of six components in commercial Roukou Wuwei pills using ultra‐high‐performance liquid chromatography with diode‐array detector

An efficient ultra‐performance liquid chromatography with diode‐array detector method was established for simultaneous determination of six active components in Roukou Wuwei pills, namely gallic acid, piperine, costundide, dehydrocostus lactone, isoalantolactone and alantolactone. Chromatographic separation of six components was successfully achieved on an Waters BEH C₁₈ column (50 × 2.1 mm, 1.7 μm) with a mobile phase composed of acetonitrile and water using a gradient elution. Gallic acid and piperine were detected at 270 nm and 343 nm, respectively; while costundide, dehydrocostus lactone, isoalantolactone and alantolactone were simultaneously measured at 225 nm. All six calibration curves showed good linearity (R² ≥ 0.9994) between the peak area of each component and corresponding concentration. Relative standard deviations for inter‐ and intra‐day precisions were <0.45 and 0.77%, respectively. The mean recovery rates ranged from 96.72 to 102.2% with relative standard deviations <2.07%. The developed method was validated in terms of linearity, precision and accuracy and then successfully applied for the quality control of commercial Roukou Wuwei samples.     

Development and validation of a rapid ultra high performance liquid chromatography with tandem mass spectrometry method for the simultaneous determination of darunavir,ritonavir, and tenofovir in human plasma: Application to human pharmacokinetics

A sensitive ultra high performance liquid chromatography with tandem mass spectrometry method was developed for the simultaneous determination of darunavir, ritonavir and tenofovir in human plasma. Sample preparation involved a simple liquid–liquid extraction using 200 μL of human plasma extracted with methyl tert‐butyl ether for three analytes and internal standard. The separation was accomplished on an Acquity UPLC BEH C₁₈ (50 mm x 2.1 mm, 1.7 μm) analytical column using gradient elution of acetonitrile/methanol (80:20, v/v) and 5.0 mM ammonium acetate containing 0.01% formic acid at a flow rate of 0.4 mL/min. The linearity of the method ranged between 20.0 and 12 000 ng/mL for darunavir, 2.0 and 2280 ng/mL for ritonavir, and 14.0 and 1600 ng/mL for tenofovir using 200 μL of plasma. The method was completely validated for its selectivity, sensitivity, linearity, precision and accuracy, recovery, matrix effect, stability, and dilution integrity. The extraction recoveries were consistent and ranged between 79.91 and 90.04% for all three analytes and internal standard. The method exhibited good intra‐day and inter‐day precision between 1.78 and 6.27%. Finally the method was successfully applied for human pharmacokinetic study in eight healthy male volunteers after the oral administration of 600 mg darunavir along with 100 mg ritonavir and 100 mg tenofovir as boosters.     

Determination of phenolic acids in olive oil by capillary electrophoresis

A CZE method for the separation and quantitation of phenolic acids (cinnamic, syringic, p-coumaric, vanillic, caffeic, 3,4-dihydroxyphenylacetic, protocatechuic), extracted from extra virgin olive oil, was developed. The sample preparation involved the LLE and SPE extraction methods. CE separation was performed in a fused silica capillary of I.D.= 50microm using as a BGE 40 mM borate buffer at pH=9.2. The separation voltage was 18kV with corresponding current of 27-28 microA. Detection was accomplished with UV-detector at lambda=200nm. The proposed method was fully validated. A good repeatability of migration time (RSD% ranged from 0.81 to 1.63) and of corrected peak area (RSD% from 2.89 to 5.77) was obtained. The linearity of detector response in the range from 5 to 50 ppm was checked, obtaining the correlation coefficient R2 values in the range: 0.9919-0.9997. Some phenolic acids in real oil samples were detected and quantified with the proposed method.     

Non-aqueous capillary electrophoresis for simultaneous separation and determination of three major active components in traditional medicinal preparations

A simple and sensitive non-aqueous capillary electrophoresis method has been developed for simultaneous assay of three bioactive components (puerarin, daidzein and wogonin) in three traditional medicinal preparations for the first time. Optimum separation of the analytes was obtained on a 47 cm x 75 microm i.d. capillary using a non-aqueous buffer system of 20% acetonitrile, 25 mm ammonium acetate and apparent pH 9.00, with applied voltage and capillary temperature of 20 kV and 16 degrees C, respectively. The relative standard deviations (RSDs) of the migration times and the peak areas of the three analytes were in the ranges 2.5--4.0% and 3.2--3.9%, respectively. Detection limits of puerarin, daidzein and wogonin were 0.090, 0.145 and 0.090 microg mL(-1), respectively. In the tested concentration range, good linear relationships (correlation coef fi cients: 0.9998 for puerarin, 0.9998 for daidzein and 0.9978 for wogonin) between peak areas and concentrations of the analytes were observed. This method has been successfully applied to simultaneous determination of the three bioactive components with recoveries from 91.0 to 114.0%.     

衍生化气相色谱-质谱法测定玩具和食品接触材料中双酚A

建立了玩具和与食品接触的材料中双酚A的衍生化气相色谱-质谱(GC-MS)检测方法。通过索氏萃取富集样品中的双酚A,与乙酸酐反应,终产物用GC-MS进行测定。优化了衍生化时间、衍生化试剂用量等衍生化条件。在最佳条件下,双酚A衍生物稳定且峰形良好,在0.05～50 mg/L范围内,相关系数(r2)在0.999以上。在0.05、1.00、10.00 mg/kg 3个添加水平下,双酚A的加标回收率为80%～93%,相对标准偏差(RSD)均小于3.7%;检出限(以信噪比(S/N)=3计)为10 μg/kg。该方法精密度好、回收率高,可用于玩具和食品接触材料中双酚A的定量检测。     

Separation and determination of B vitamins and essential amino acids in health drinks by CE-LIF with simultaneous derivatization

An efficient and sensitive method for the separation and determination of three essential amino acids and three B vitamins by CE-LIF with a simultaneous derivatization procedure was developed. The conditions for derivatization and separation of these micronutrients were investigated. FITC was used as the reagent for fluorescence tagging of arginine (Arg), valine (Val), tryptophan (Trp), folic acid (FA), and niacinamide (NA). Riboflavin (RF) was detected without derivatization. Derivatization of analytes dissolved in borate solution was performed by successive introduction of fluorescence reagent and analytes followed by water bathing at 43°C. The molar ratio of sample/reagent (S/R), derivatization temperature, and incubation time significantly influenced the efficiency of derivatization. To maximize the fluorescence yield, a high S/R (≥20) was required. The nonderivatized RF and five derivatized analytes were separated in the optimized CE-LIF system with the application of 22 kV voltage and 25 mM borate buffer at pH 9.85. Validation of the method showed good linearity for the corrected peak areas versus standard concentrations for the six analytes. The RSDs (n = 3) of the migration time and the peak area obtained for the analytes ranged from 0.4 to 1.1% and from 1.9 to 4.4%, respectively. The developed method, with the lowest LOD of 0.5 nM, was successfully applied for the efficient derivatization and determination of B vitamins in four health drink samples.     

Analysis of acetylcholine and choline in microdialysis samples by liquid chromatography/tandem mass spectrometry

A sensitive liquid chromatography/electrospray ionisation tandem mass spectrometric (LC/ESI-MS/MS) method was developed for the analysis of acetylcholine and choline in microdialysis samples. A Ringer's solution that contains high (150 mM) concentrations of inorganic salts was used to extract acetylcholine and choline from a rat or mouse brain. The separation of acetylcholine, choline, an internal standard acetyl-beta-methylcholine, endogenous compounds and inorganic cations was achieved with hydrophilic interaction chromatography using a diol column. The eluent consisted of 20 mM ammonium formate (pH 3.3) and acetonitrile (20:80) which is favourable for the ESI process. Limits of detection (signal-to-noise (S/N) ratio = 3) of 0.02 nM (0.2 fmol) for acetylcholine and 1 nM (10 fmol) for choline were observed using standards diluted in Ringer's solution. A good linearity was obtained from the limit of quantitation: 0.1 nM (S/N ratio = 10) to 50 nM (r = 0.999) for acetylcholine and within the concentration range of 100-3500 nM (r = 0.998) for choline. The between-day repeatability of the method was good; RSD was 3.1% at 1 nM level of acetylcholine and 3.5% at 1000 nM level of choline. The recoveries for addition of 1 or 2.5 nM acetylcholine and 0.2 or 1 microM choline in microdialysis balancing samples were between 93 and 101% indicating that no suppressing endogenous compounds were co-eluting with acetylcholine or choline. The developed method was applied to the analysis of microdialysis balancing samples collected from rat and mouse brains.     

Study of the enantioseparation of imazaquin and enantioselective degradation in field soils by CZE

A fast, simple, and sensitive CE method was developed to study the separation and degradation of imazaquin enantiomers in field soils. The parameters pH and concentration of the buffer electrolyte, type and concentration of the chiral selectors, applied voltage, and temperature of the CE system were investigated. Sodium hydrogen phosphate (50 mM) at pH 10.1 containing 30 mM hydroxypropyl-beta-CD (HP-beta-CD) was found to be the suitable BGE. The reliable determination for imazaquin enantiomers was obtained at 20 kV applied voltage, 15 degrees C separation system, and detection at 214 nm. Under the optimal conditions, the LODs (S/N = 3) were 0.0097 and 0.0098 mg/kg; linearity ranged from 0.019 to 1.24 and 0.020 to 1.26 mg/kg for the first-eluting peak of imazaquin (imazaquin-I) and the second-eluting peak of imazaquin (imazaquin-II) in soil, respectively. The precision in terms of the percentage of RSD (%RSD) calculated from peak area was not greater than 5%. With the suitable characteristics of the present method, it was applied to the study of enantioselective degradation in field soil. According to the calculated values of the enantiomer ratio (ER) and the rate constant of degradation (k), the results suggested that the degradation rate of the two enantiomers were slightly different, and that the pH of the soil had a strong influence on the rate of degradation.     

Determination of ascorbic acid content of some fruit juices and wine by voltammetry performed at pt and carbon paste electrodes

A method was developed for assessing ascorbic acid concentration in fruit juices and wine by differential pulse voltammetry. The oxidation peak for ascorbic acid occurs at about 530 mV (versus SCE) on a Pt strip working electrode and at about 470 mV on a carbon paste working electrode. The influence of the operational parameters like the pulse amplitude and the pulse period on the analytical signal was investigated. The obtained calibration graph shows a linear dependence between the peak height and ascorbic acid concentration within the range 0.31-20 mM with a Pt working electrode, and within the range 0.07-20 mM with a carbon paste working electrode. The equation of the calibration graph was y = 21.839x + 35.726, r2 = 0.9940, when a Pt strip electrode was used (where y represents the value of the current intensity measured for the peak height, expressed as μA and x the analyte concentration, as mM). R.S.D. = 2.09%, n = 10, C(ascorbic acid) = 2.5 mM. The equation of the calibration graph was y = 3.4429x + 5.7334, r2 = 0.9971, when a carbon paste electrode was used (where y represents the value of intensity measured for the peak height, expressed as μA and x the analyte concentration, as mM). R.S.D. = 2.35%, n = 10, C(ascorbic acid) = 2.5 mM. The developed method was applied to ascorbic acid assessment in fruit juices and wine. The ascorbic acid content determined ranged between 6.83 mg/100 mL juice for soft drinks (Fanta Madness) and 54.74 mg/100 mL for citrus (lemon) juices obtained by squeezing fruit. Different ascorbic acid concentrations (from standard solutions) were added to the analysed samples, the degree of recovery being comprised between 94.74 and 104.97%. The results of ascorbic acid assessment by differential pulse voltammetry were compared with those obtained by cyclic voltammetry. The results obtained by the two methods were in good agreement.     

Direct analysis of formate in human plasma,serum and whole blood by in-line coupling of microdialysis to capillary electrophoresis for rapid diagnosis of methanol poisoning

A simple method using direct injection of human blood samples and quantitative analysis of formate was developed for rapid diagnosis of methanol poisoning. A sample pretreatment device including a 500 Da molecular weight cut-off dialysis membrane was in-line coupled to capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-C⁴D). 50 μL of 1:9 diluted blood samples and 50 μL of DI water were filled into the donor and the acceptor chamber, respectively, and small ionic species in blood samples were electrokinetically injected across the dialysis membrane directly into the separation capillary. Matrix components, such as red blood cells, proteins, lipids and other high molecular weight compounds, were retained by the dialysis membrane and did not interfere with subsequent CE separation. Formate was separated from other small anions in an optimized background electrolyte solution consisting of 20 mM l-histidine and 25 mM l-glutamic acid at pH 4.8. The method showed excellent analytical parameters in terms of repeatability and linearity; RSD values for migration times and peak areas at a formate concentration typical for methanol poisoning were below 0.3% and 7.4%, respectively, and linear calibration curves with correlation coefficients better than 0.999 were obtained. Limit of detection and limit of quantification were 15 and 50 μM formate in original (undiluted) blood samples, respectively. The method was applied to determination of formate in serum samples of a patient diagnosed with acute methanol poisoning.     

Development and validation of a capillary electrophoresis method with capacitively coupled contactless conductivity detection (CE-C(4) D) for the analysis of amikacin and its related substances

Amikacin is a semisynthetic aminoglycoside antibiotic derived from kanamycin A that lacks a strong UV absorbing chromophore or fluorophore. Due to the physicochemical properties of amikacin and its related substances, CE in combination with capacitively coupled contactless conductivity detection (CE-C(4) D) was chosen. The optimized separation method uses a BGE composed of 20 mM MES adjusted to pH 6.6 by l-histidine and 0.3 mM CTAB that was added as flow modifier in a concentration below the CMC. Ammonium acetate 20 mg.L(-1) was used as internal standard. 30 kV was applied in reverse polarity on a fused silica capillary (73/48 cm; 75 μm id). The optimized separation was obtained in less than 6 min with good linearity (R(2) = 0.9996) for amikacin base. It shows a good precision expressed as RSD on relative peak areas equal to 0.1 and 0.7% for intraday and interday, respectively. The LOD and LOQ are 0.5 mg.L(-1) and 1.7 mg.L(-1) , respectively.