SPE and large-volume sample stacking in MEKC for determination of doxycycline in biological fluids: comparison of direct injection to SPE-MEKC

A novel and simple method has been developed for the determination of doxycycline (DOX) in biological fluids. The method is based on SPE, large-volume sample stacking (LVSS) and MEKC with UV-DAD detection. Six SPE cartridges have been used in investigation for sample clean up and pre-concentration (Supelco LC-8, LC-18, LC-SCX, and LC-WCX, as well as Strata-X and X-C). DOX was determined on a 56 cm (effective length 50 cm) x 50 microm id fused-silica capillary. The BGE was 20 mM borate buffer, pH 9.3, containing 80 mM SDS and 7.5% v/v of methanol (30 sx50 mbar), and the temperature and voltage were 25 degrees C and 30 kV, respectively. The analytical wavelength was set at 210 nm. Under optimized conditions it is possible to determine DOX in human serum, urine, semen, tears and saliva with recovery of 97.5% (RSD 2.5%). The method was shown to be sensitive (LOD is 1 microg/L) and precise (intra-day RSD 0.2 and 2.4%; inter-days 0.4 and 3.5% for migration time and peak area, respectively). Results for developed SPE-LVSS-MEKC were compared with LVSS-MEKC method with direct sample injection. The new LVSS-MEKC method is presented as a useful technique for rapid determination without extraction procedure of DOX in human urine and serum, using 80 mM of SDS, 10% v/v of methanol and 40 mM borate buffer (pH 9.3; 30 s x 50 mbar; 25 degrees C; 30 kV; 350 nm), but not for the other biological fluids, according to lower sensitivity of the method and because of the sample composition.     

Spectrofluorimetric determination of moxifloxacin in tablets, human urine and serum

A spectrofluorimetric method to determine the antibiotic moxifloxacin is proposed and was applied to pharmaceuticals, human urine and serum. The fluorimetric method allows the determination of 30-300 ng mL-1 moxifloxacin in aqueous solution containing phosphoric acid-phosphate buffer (pH 8.3) with lambda exc = 287 nm and lambda em = 465 nm. Detection and quantification limits were 10 and 30 ng mL-1, respectively, with a relative standard deviation (n = 10) of 2%. This method was applied to the determination of moxifloxacin in three Spanish commercial pharmaceutical formulations. Another variant of the method in micellar medium allows the direct measurement of moxifloxacin in human serum and urine by standard additions. The enhanced fluorescence of moxifloxacin in 8 mM sodium dodecyl sulfate (SDS) solution at pH 4.0 (acetic acid-acetate buffer) for lambda exc = 294 nm and lambda em = 503 nm shows the same linear range as the aqueous method with a 25% lower slope (with detection and quantification limits of 15 and 60 ng mL-1, respectively, and a relative standard deviation of 1.3%), but permits the background fluorescence for urine and serum blanks to be minimized. Hence, sufficient sensitivity is reached to determine therapeutic concentrations of the drug in urine (average recovery 102 +/- 2%) and serum (average recovery 105 +/- 2%) samples.     

Fluorescence and terbium-sensitised luminescence determination of garenoxacin in human urine and serum

Fluorescence and terbium-sensitised luminescence properties of new quinolone garenoxacin have been studied. The fluorimetric method allows the determination of 0.060-0.600 μg ml⁻¹ of garenoxacin in aqueous solution containing HCl/KCl buffer (pH 1.5) with λ_exc=282 nm and λ_em=421 nm. Micellar-enhanced fluorescence was also studied, leading to a higher than 400% increase in analytical signal in presence of 12 mM sodium dodecyl sulphate (SDS), allowing the determination of 0.020-0.750 μg ml⁻¹ of garenoxacin. The terbium-sensitised luminescence method allows the determination of 0.100-1.500 μg ml⁻¹ of garenoxacin in 12 mM SDS solution containing 0.08 M acetic acid/sodium acetate buffer (pH 4.1) and 7.5 mM Na₂SO₃ (chemical deoxygenation agent), with λ_exc=281 nm and λ_em=546 nm. Relative standard deviation (R.S.D.) values for the three methods were in the range 1.0-2.0%. The proposed procedures have been applied to the determination of garenoxacin in spiked human urine and serum.     

Flow-injection chemiluminescence determination of ofloxacin and levofloxacin in pharmaceutical preparations and biological fluids.

A novel, rapid and sensitive analytical method is described for determination of ofloxacin and levofloxacin by enhanced chemiluminescence (CL) with flow-injection sampling. The method is based on the CL reaction of the Ce(IV)-Na2S2O4-ofloxacin/levofloxacin-H2SO2 system. The enhanced CL mechanism was developed and the optimum conditions for CL emission were investigated. The CL intensity was correlated linearly (r = 0.9988) with the concentration of ofloxacin (or levofloxacin) in the range of 1.0 x 10(-8) - 1.0 x 10(-7) g ml(-1) and 1.0 x 10(-7) - 6.0 x 10(-6) g ml(-1). The detection limit (S/N = 3) is 7 x 10(-9) g ml(-1). The relative standard derivation (RSD, n = 11) is 2.0% for ofloxacin at 4 x 10(-7) g ml(-1) and for levofloxacin at 6 x 10(-7) g ml(-1). This method has been successfully applied for the determination of ofloxacin and levofloxacin in pharmaceutical preparations and biological fluids with satisfactory results.     

Spectrophotometric determination of sulfur dioxide in air, using thymol blue

A simple and sensitive spectrophotometric method was developed for the determination of trace amounts of sulfur dioxide. The method is based on the reaction of SO2 with a known excess of ICI as the oxidant. The unreacted ICI iodinates thymol blue under acidic conditions. The lambdamax of thymol blue is at 545 nm under acidic conditions, and on lodination lambdamax shifts to 430 nm. This shift results in a decrease in the absorbance at 545 nm. The amount of uniodinated thymol blue present depends on the concentration of unreacted ICI, which in turn depends on the SO2 concentration. The system obeys Beer's law in the range 0-30 microg SO2 in a final volume of 25 mL, having a molar absorptivity of 3.2 x 10(4) L/mol cm with a relative standard deviation (RSD) of 2% at 24 microg SO2 (n = 10). The uniodinated dye can be extracted into 5 mL isoamyl alcohol under acidic conditions for measurement of absorbance. The extraction method obeys Beer's law in the range 0-5 microg SO2, having a molar absorpitivity of 4.16 x 10(4) L/mol x cm with an RSD of 1.9% at 4 microg SO2 (n = 10). The method has been successfully applied to the determination of atmospheric SO2.     

Determination of mannitol and sorbitol in infusion solutions by capillary zone electrophoresis using on-column complexation with borate and indirect spectrophotometric detection

Capillary zone electrophoresis with indirect UV detection at 215 nm was applied for the separation and determination of mannitol (MA), sorbitol (SO) and xylitol in the form of anionic borate-polyol complexes. The separation was carried out in a fused silica capillary (total length 60 cm, effective length 50 cm, I.D. 50 microm) at 25 kV. The optimized background electrolyte was 200 mM borate buffer (pH 9.3, adjusted with triethylamine) containing 10 mM 3-nitrobenzoate as the chromogenic co-ion. The separation took approximately 13 min. The rectilinear calibration range was 0.2-2 mg mL(-1) for MA and SO when using xylitol (1 mg mL(-1)) as the internal standard. The limit of detection at a S/N of 3 was approximately 30 microg mL(-1) for either analyte. The method was used for the assay of MA or SO in pharmaceutical infusion solutions. The RSD values were 0.15% or 1.07% (n=6) when determining 100 mg mL(-1) of MA or 50 mg mL(-1) of SO in commercial infusion solutions. The results were in good agreement with those of pharmacopoeial iodimetric titration.     

Determination of aristolochic acid in Chinese herbal medicine by capillary electrophoresis with laser-induced fluorescence detection

We have demonstrated the analysis of aristolochic acids (AAs) that are naturally occurring nephrotoxin and carcinogen by capillary electrophoresis in conjunction with laser-induced fluorescence detection (CE-LIF). Owing to lack of intrinsic fluorescence characteristics of oxidized AAs (OAAs), reduction of the analytes by iron powder in 10.0 mM HCl is required prior to CE analysis. The reduced AAs (RAAs) exhibit fluorescence at 477 nm when excited at 405 nm using a solid-state blue laser. By using 50.0 mM sodium tetraborate (pH 9.0) containing 10.0 mM SDS, the determination of AA-I and AA-II by CE-LIF has been achieved within 12 min. The CE-LIF provides the LODs of 8.2 and 5.4 nM for AA-I and AA-II, respectively. The simple CE-LIF method has been validated by the analysis of 61 Chinese herbal samples. Prior to CE analysis, OAAs were extracted by using 5.0 mL MeOH, and then the extracts were subjected to centrifugation at 3,000 rpm for 5 min. After reduction, extraction, and centrifugation, the supernatants were collected and subjected to CE analysis. Of the 61 samples, 14 samples contain AA-I and AA-II, as well as 10 samples contain either AAI or AAII. The relative standard deviation (RSD) values of the migration times for AA-I and AA-II are less than 2.5% and 2.1% for three consecutive measurements of each sample. The RSD values for the peak heights corresponding to AA-I and AA-II in most samples are about 8.0% and 10.0%, respectively. The result shows that the present CE-LIF approach is sensitive, simple, efficient, and accurate for the determination of AAs in real samples.     

Flow-injection spectrophotometric determination of novalgin in pharmaceuticals using micellar medium.

A sensitive flow-injection (FI) procedure with spectrophotometric detection in a micellar medium is proposed for the determination of novalgin. The method is based on the instantaneous formation of a red-orange product (lambda(max) = 510 nm) after the reaction between novalgin and p-dimethylaminocinnamaldehyde (p-DAC) in a dilute acid medium. The sensitivity of this reaction was increased by a factor of 5.6 in the presence of sodium dodecyl sulfate (SDS). Experimental design methodologies were used to optimize the chemical and FI variables. The calibration curve was linear in the range of 1.45 x 10(-6) to 2.90 x 10(-5) mol L(-1) with an excellent correlation coefficient (r = 0.9999). The detection limit was 1.31 x 10(-7) mol L(-1) (n = 20, RSD = 2.0%). No interferences were observed from the common excipients. The results obtained by the proposed method were favorably compared with those given by the iodometric reference method at 95% confidence level.     

Simultaneous micellar liquid chromatographic analysis of seven water-soluble vitamins: optimization using super-modified simplex

A super-modified simplex (SMS) method has been used to optimize the mobile phase used for separation of seven water-soluble vitamins in multivitamin tablets by gradient micellar liquid chromatography (MLC) with ultraviolet (UV) detection at 254, 295, and 361 nm. Effect of column temperature and addition of organic modifier to the mobile phase on separation efficiency were investigated: the appropriate conditions used were a temperature of 35 degrees C and 1-butanol modifier. The sodium dodecyl sulfate (SDS) concentration, pH, and 1-butanol% in the mobile phase were chosen for simultaneous optimization using the SMS method. The optimum mobile phase was found to be 16 mmol L(-1) (mM) SDS, 0.02 M phosphate buffer, pH 3.6, and a gradient of 3.5-10% (v/v) butanol. The total analysis time for vitamins was 75 min. The analytical parameters including linearity ( r>0.9970), limit of detection (0.12-50 micro g mL(-1)), precision of method (relative standard deviation (RSD) <8.90%), and accuracy obtained by the recovery assay (88-103%) support the usefulness of the proposed method for the determination of the water-soluble vitamins.     

Enhanced detection of porphyrins by capillary electrophoresis-laser induced fluorescence

A highly sensitive technique for the analysis of urinary porphyrins using capillary electrophoresis (CE) coupled with laser-induced fluorescence (LIF) detection is reported. Separation of mesoporphyrin IX, coproporphyrin, uroporphyrin and the penta-, hexa- and heptacarboxylic acid porphyrins was achieved in 11 min using a 10 mM 2-(N-cyclohexylamino)ethanesulfonic acid (CHES, pH 10) -75 mM sodium dodecyl sulfate (SDS) buffer. Migration time and peak area repeatability were less than 1 and 5% relative standard deviation (RSD), respectively. Limits of detection of 20 pM (2 x 10(-11) M) were achieved employing the recently introduced Nichia violet diode laser for excitation at 400 nm. This represents an enhancement in sensitivity of over two orders of magnitude compared to previous reports. This high sensitivity for urinary porphyrins was demonstrated through the quantification of coproporphyrin and uroporphyrin in urine samples after up to a 100-fold dilution.     

Separation and determination of flavonoids using microemulsion EKC with electrochemical detection

A selective and sensitive method of microemulsion EKC (MEEKC) with electrochemical detection (ED) was developed for separation and determination of 14 flavonoids. In order to obtain the better stability for the studied flavonoids, oil (ethyl acetate) with low interfacial surface tension was employed as organic solvent. A running buffer composed of 0.9% (w/v, 30 mM) SDS, 0.9% (w/v, 21 mM) sodium cholate (SC), 0.9% (w/v, 121 mM) butan-1-ol, 0.6% (w/v, 68 mM) ethyl acetate, and 98.2% v/v 10 mM Na(2)B(4)O(7)-20 mM H(3)BO(3) buffer (pH 7.5) was applied for the separation of flavonoids. Under the optimum conditions, the relationship between peak currents and analyte concentrations was linear over about 1.3 and 1.7 orders of magnitude with detection limits (defined as S/N = 3) ranging from 0.02 to 0.5 microg/mL for all analytes. This method was applied for the determination of flavonoids in real samples with simple extraction procedures, and the assay results were satisfactory.     

A novel ion chromatographic method based on cation-exchange and acid-base interactions for the simultaneous determination of total alkalinity and monovalent cations in samples of microliter volume

An ion chromatographic (IC) method based on the use of titrant (strong acid) as the stationary phase was developed for simultaneous determination of total alkalinity (TA) and monovalent cations. The titrant used in this study was obtained by initially loading lithium dodecylsulfate (Li-DS) onto a reversed-phase material and then conditioning the column with a slightly acidified aqueous LiCl solution (a mixture of 50.0 mM LiCl and 0.1 mM H2SO4). When a small amount of a basic sample was injected onto a column prepared in this way, the basic species (Bn-) reacted predominantly with H+ on the stationary phase and the reaction with the eluent phase was negligible due to the very low concentration of eluent H+ (in the eluent, a molar ratio of [Li+]/[H+] = 250:1 applied). The stationary phase H+ consumed in the acid-base reaction was then re-supplied by H+ from the eluent. By monitoring the conductance of the eluent using conductivity, an induced peak resulting from the basic species was observed. Calibration graphs of peak areas vs. molar concentration of the basic species for OH-, HCO3- and H2PO4- were found to be identical. CO3(2-), HPO4(2-), and B4O7(2-) also gave identical calibration curves but their slope values were twice those for HCO3-. The detection limit for HCO3- was less than 3.2 microM and the calibration curve was linear up to 12.3 mM (injection volume, 100 microL). Seawater was directly analyzed and its total alkalinity was found to be 2.87 mM (RSD 0.53%, n = 5), which was in good agreement with the result of 2.88 mM (RSD 3.2%, n = 5) obtained using auto-potentiometric titration. Na+ and K+ were determined simultaneously and the concentrations were 481.6 and 10.6 mM, respectively.     

胶束扫集毛细管电泳快速测定止咳露中的麻黄碱和可待因

采用胶束扫集毛细管电泳, 建立了快速测定止咳露中麻黄碱和可待因含量的方法, 并通过日间实验、柱间实验等对方法的稳定性进行了考察研究.胶束扫集电动色谱缓冲体系含60 mmol/L 十二烷基磺酸钠, 10 mmol/L NaH2PO4 (pH 2.20), 18%乙腈(V/V), 分离电压-14 kV, 测量波长200 nm. 讨论了pH、 SDS浓度、样品溶剂等对分离效果的影响. 在优化条件下, 麻黄碱和可待因均在5 min内出峰, 方法检出限(μg/mL)、线性范围(μg/mL)、相关系数分别为: 麻黄碱 0.433、 1.73～27.7、 0.9997, 可待因0.833、 3.33～50.3、 0.9996, 回收率在96.7%～103.5%之间. 峰面积日内RSD≤4.2% (n=5), 日间RSD≤8.0% (n=5), 柱间实验RSD≤2.3% (n=3).     

Micellar electrokinetic capillary chromatography determination of zinc bacitracin and nystatin in animal feed

An MEKC procedure was developed for the separation of zinc bacitracin (Zn-BC) and nystatin (NYS) in mixtures and in animal feedstuff. The running buffer was 15 mM borate/19 mM phosphate, pH 8.2, containing 20 mM SDS and 10% v/v methanol. Samples were run at 25 degrees C, the applied voltage was 25 kV, and an additional pressure of 5 mbar was applied. Both analytes were detected by UV simultaneously at 215 nm, Zn-BC alone at 192 and 254 nm, and NYS alone at 305 nm. The method was shown to be specific, accurate (recoveries were 100.0 +/- 0.6% and 100.1 +/- 0.6% for Zn-BC and NYS, respectively), linear over the tested range (correlation coefficients 0.9991 and 0.9994), and precise (RSD below 1.3% for both analytes). The method was applied to determine Zn-BC and NYS as additives in animal feed.     

Determination of doxycycline in pharmaceuticals and human urine by micellar electrokinetic capillary chromatography

Micellar electrokinetic capillary chromatography (MEKC) was performed at 25 °C and 30 kV (under a pressure of 15 mbar), using 30 mM borate buffer containing 60 mM sodium dodecysulfate (SDS) and 5% (v/v) methanol as background electrolyte (pH 9.0) to determine doxycycline. UV detection was at 350 nm. The method was shown to be specific, accurate (recovery was 100.3 ± 1.0%), linear over the tested range (correlation coefficient 0.9995) and precise (RSD <1.9%). The method was used to determine doxycycline in tablets, capsules and human urine after oral application.     

Synthesis of meso-tetra-(3,5-dibromo-4-hydroxylphenyl)-porphyrin and its application to second-derivative spectrophotometric determination of lead in clinical samples

A new very sensitive and selective chromogenic reagent, meso-tetra-(3,5-dibromo-4-hydroxylphenyl)porphyrin [T(DBHP)P], was synthesized and studied for the determination of trace lead in detail. In 0.10 mol l-1 NaOH medium, lead reacts with T(DBHP)P to form a 1:2 yellow complex, which gives a maximum absorption at 479 nm; 0-0.48 microgram ml-1 Pb(II) obeyed Beer's law. The molar absorptivity of the complex and Sandell's sensitivity are 2.5 x 10(5) 1 mol-1 cm-1 and 0.000812 microgram cm-2, respectively. Second-derivative spectrophotometry is better than conventional spectrophotometry in sensitivity and selectivity, and its limit of quantification, limit of detection and relative standard deviation are 0.70 ng ml-1, 0.21 ng ml-1 and 1.0%, respectively. Ca (3250-fold), Mg (2000-fold), Sr (1000-fold), Ba (750-fold), Al (1000-fold), Bi (500-fold), Fe (2000-fold), Co (750-fold), Ni (1000-fold), Cu (750-fold), Zn (1250-fold), Cd (2500-fold) and Ag (550-fold) do not interfere with the determination of lead. The chromogenic system is remarkably superior to other reagents, especially porphyrin compounds. The influence caused by oxygen in air or in solution can be easily eliminated by adding Na2SO3. The reaction is very stable, the stability constant of the complex being 1.2 x 10(45). The chromogenic reaction is completed within 1 min at room temperature when 8-hydroxylquinoline is used as catalyst. The proposed method has been applied to the direct determination of trace lead in clinical samples. The accuracy and precision are both very satisfactory.     

Determination of oxoanions in river water by capillary electrophoresis

A new capillary electrophoresis (CE) procedure was developed for simultaneous determination of ten oxoanions (CrO4(2-), SeO4(2-), MoO4(2-), WO4(2-), VO4(3-), SeO3(2-), As04(3-), TeO3(2-), TeO4(2-), and AsO3(3-)) which were baseline-separated from each other and from the interfering UV absorbing anions (NO3- and NO2-) commonly found in environmental water samples. The new background electrolyte system developed contained 5 mM potassium phosphate and 0.007 mM octadecyltrimethylammonium hydroxide, pH 11.2. The optimized working conditions were electrokinetic sampling at -5 kV for 10 s, running voltage at -15 kV with 5 microA current, and detection wavelength at 205 nm. No interference was observed for non-UV-absorbing anions and UV-absorbing anions up to 20 and 10 times higher concentrations respectively. The speed of analysis was fast, with a complete CE run within 6 min. Wide linear ranges (1-2,000 microg/L), good repeatability in migration time (relative standard deviation RSD 0.55-2.8%), satisfactory precision in peak area (RSD 3.8-5.6%) and peak height (RSD 3.9-5.3%) measurement, and detection limits (1-25 microg/L) sufficiently sensitive to detect oxoanions found in environmental water samples were obtained. The reliability of the CE procedure developed had been established by recovery test and parallel method determination using atomic absoprtion spectrophotometry for real river water sample.     

胶束毛细管电泳在线推扫技术分离检测猪肉组织中痕量喹诺酮类药物

利用胶束毛细管电泳法结合在线推扫富集技术对组织中残留的痕量环丙沙星、氧氟沙星和恩诺沙星进行了检测, 弥补了毛细管电泳检测灵敏度低的缺点, 大大减化了操作过程, 为动物食品组织中残留的痕量药物检测提供了一种新的简便可靠的方法.     

On-line concentration by field-enhanced sample injection with reverse migrating micelles in micellar electrokinetic capillary chromatography for the analysis of flavonoids in Epimedium brevicornum Maxim

A simple and sensitive micellar electrokinetic capillary chromatography (MEKC) method was developed for the separation and determination of six flavonoids in Epimedium brevicornum Maxim. Field-enhanced sample injection with reverse migrating micelles (FESI-RMM) was used for on-line concentration of the flavonoids. An electrolyte containing 20 mM H3PO4, 100 mM SDS, 20% acetonitrile and 2% 2-propanol (pH 2.0) was chosen as the electrophoretic buffer. By optimizing the stacking conditions, about 40-360-fold improvement in the detection sensitivity was obtained for the flavonoids.     

Determination of primary biogenic amines in various food products using isocratic HPLC with 3-(4-chlorobenzoyl)-quinoline-2-carboxaldehyde as a new precolumn fluorogenic reagent

A simple HPLC approach has been successfully established for the sensitive determination of six biogenic amines (BAs) in food samples. The method involves derivatization with 3-(4-chlorobenzoyl)-quinoline-2-carboxaldehyde newly synthesized as a new fluorogenic reagent followed by LC isocratic elution mode. The optimization of both derivatization and separation conditions is carefully studied. Related analyses of the eluted compounds, in the presence of MeOH/THF/H(2)O (78:2.5:19.5 v/v/v) as a mobile phase containing 8 mM, pH 6.0 phosphate buffer solution, have been carried out on a C(18) column. The LOD (S/N = 3) of 2.5 nM, RSD value from 1.0 to 5.1% in peak areas, and good response linearity (R(2) >0.9936) are provided with fluorescence detection at lambda(ex)/lambda(em) = 480/545 nm. Obviously, recovery ranging from 95 to 107% in this method demonstrates its accuracy for determination of histamine, tyramine, 2-phenylethylamine, putrescine, cadaverine, and spermidine in the storage fish sample. Thus, the present method could be developed to monitor BAs in fish, cucumber, and spinach samples.