Development and Validation of Gel-Chromatographic and Spectrophotometric Methods for Quantitative

Acta Chromatographica 22(2010)3, 375–390 DOI: 10.1556/AChrom.22.2010.3.30231–2522 © 2010 Akadémiai Kiadó, BudapestDevelopment and Validation of Gel-Chromatographic and Spectrophotometric Methods for Quantitative Analysis of Bioactive Copper Complexes in New Antihypocupremical FormulationsI. S AVIC *, G. N IKOLIC , I. S AVIC , AND M. C AKICDepartment of Pharmaceutics, Faculty of Technology, University of Nis,Bulevar Oslobodjenja 124, Leskovac 16000, SerbiaE-mail: ici86@info-net.rs or ici_teh@Summary. Gel-permeation chromatographic (GPC) and visible spectrophotometric methods have been developed and validated for quantitative analysis of complexes of copper(II) with the polysaccharides pullulan and dextran, active pharmaceutical com-pounds in new antihypocupremical formulations. Linearity, precision, accuracy, speci-ficity, and limits of detection (LOD) and quantification (LOQ) were determined in ac-cordance with ICH Q2(R1) guidelines. GPC was performed isocratically with redistilled water as mobile phase at a flow rate of 1 mL min −1. Visible spectrophotometry was per-formed in water, using 640 nm for direct assay of the copper(II) complex with pullulan and dextran. The calculated F and t values at the 95% confidence level were less than the theoretical values, showing there were no significant differences between the perform-ance of the methods.Key Words: antihypocupremical formulations, GPC, visible spectrophotometryIntroductionMicrocytic hypochromic anemia is one of the outcomes of copper defi-ciency. Many preparations are used for treatment. These are mixtures of mi-croelements in the form of their salts (CuSO 4 or CuCl 2), which are toxic and unstable [1, 2]. Copper complexes with amino acids can also be used for treatment, as has been well described in the literature [3]. The active phar-maceutical compound has a repetitive dose schedule (0.6–2 mg daily) and is absorbed from the lower part of the gastrointestinal tract.M etal complexes with polysaccharides and their derivatives are of growing importance in medicine and pharmacy. For example, Polypher is well known as a blood substitute [4]. New blood substitutes with hemo-I. Savic et al. 376stimulating and antianemic function, complexes of dextran and pullulan with Fe(II), Fe(III), Cu(II), and Co(II) ions, differ from the existing analogues in having good biocompatibility and hemocompatibility and more pro-nounced and prolonged action [5–8]. Magnetic complexes based on poly-saccharide derivatives with Fe, Ca, Zn, Co, Ni, and Cu oxides are used in roentgenology studies. These complexes must be very stable during pro-longed storage and must not be toxic [9, 10].Fig. 1. Structural models of the complexes of Cu(II) with dextran (a) and pullulan (b)Reduced low-molar pullulan (RLMP,Мw6000 g mol−1) and reduced low-molar dextran (RLMD, Мw 5000 g mol−1) have been chosen as new ma-terials for complexing and subsequent interaction with Cu(II) ions. In alka-line solutions (pH > 7), Cu(II) ions form complexes with these ligands [11, 12]. The bioactive complex of copper(II) with RLMP contains 13.1% copper and the complex of copper(II) with RLMD contains 19.8% copper. The com-plexes obtained are green, amorphous, almost odorless and freely soluble in water at 25°С [13].In the solid state the complexes are very stable during prolonged storage at room temperature and are not toxic [14, 15]. The struc-Quantitative Analysis of Bioactive Copper Complexes 377 tures of bioactive copper(II) complexes with the oligosaccharides dextran or pullulan (Fig. 1) were confirmed by physicochemical and spectroscopic characterization [16–19].Bioactive copper(II) complexes with RLMP and RLMD, as active phar-maceutical compounds, were prepared by making solid dosage forms (tab-lets) in the laboratory. These complexes are not yet official in any pharma-copoeia. The patent literature has data on the synthetic procedure only, with physicochemical and spectroscopic characterization [11, 20]. Results from determination of correlations between the structure and stability of complexes of copper(II) with RLMD by use of conductiometry have been well described in the literature [15, 21].F or routine analysis of the active drug content of pharmaceutical for-mulations we developed simple, specific, and direct GPC and visible spec-trophotometric methods with sufficient reliability. Both methods fulfilled European Pharmacopoeia analytical quality requirements [22] for content uniformity testing of finished pharmaceutical products when these are pre-sent as single active principles, and, hence, can be successfully used for rou-tine quality control of tablets.ExperimentalMaterialsThe bioactive copper(II) complex with dextran oligosaccharides was syn-thesized by the original procedure [12]. Synthesis of the complex of cop-per(II) with pullulan oligosaccharides has been described in detail by Niko-lic et al. [11]. Antihypocupremical tablets [F1, which contains 1.5 mg cop-per(II) complex with pullulan, and F2, which contains 1.5 mg copper(II) complex with dextran] were prepared by the original procedure [23].Qual-ity-control tests for measurement of friability, disintegration, hardness, weight variation, and assay were performed on all formulations in accor-dance with the European Pharmacopoeia [22].Extraction of the Active Ingredient from FormulationsThe tablets were accurately weighed and powdered. The amount of drug in the weighed quantity of powder was calculated on the basis of the label claim and the active ingredients in formulations F1 and F2 were then ex-tracted with water. The solutions were sonicated for 20 min and then fil-tered through Whatman no. 1 filter paper into 25-mL volumetric flasks.I. Savic et al. 378Appropriate dilutions were prepared and the solutions were subjected for both GPC and visible spectrophotometric analysis.Instrumentation and Analytical ConditionsThe validation runs were performed on two HPLC instruments. The first was an Agilent Technologies (Palo Alto, CA, USA) 1100 liquid chromato-graph with a Zorbax Eclipse XDB PSM-60 column (Eldex Laboratories, Napa, CA, USA) and Waters (Milford, MA, USA) 2414 refractive index (RI) detector. The second system was a Hitachi (San Jose, CA, USA) L-7100 high-pressure pump, Waters 717 plus autosampler, Eldex CH-150 column, and Waters 410 RI detector. Data acquisition and processing were performed us-ing AgilentChemStation automation system software. GPC analysis was performed by isocratic elution with water at a flow rate of 1 mL min−1. The water used as mobile phase was prepared freshly and filtered through a 0.45-μm membrane filter (Millipore, USA). Injection volume was 80 μL. Quantification was effected by measurement of absorption at 640 nm.V alidation runs were performed on two double-beam visible spectro-photometer: a Varian Cary-100 Conc and a Perkin–Elmer (Germany) Lambda 15. The aqueous solutions were measured in 1-cm quartz cells. The spectra were recorded using a 1-mm slit and 120 nm min−1 scanning speed. The assay was performed at the analytical wavelength 640 nm for formula-tions F1 and F2.A Mettler–Toledo AG245 electronic balance, an Electrolab Friabilator USP (XXIII), an Electrolab disintegration tester (USP), and an Erweka TBH20 (hardness tester) were also used for performing the quality-control tests on the antihypocupremical formulations.Preparation of Standard Solutions and Quality-ControlStandard SolutionsGPC MethodPrimary stock solutions of copper(II) complex with polysaccharides pullu-lan or dextran (1 mg mL−1) were prepared in ultra-pure water and further diluted with water to obtain calibration standards in the concentration range 18–180 μg mL−1. Quality-control (QC) standard solutions were run with each batch of calibration standards to calculate validation data. QC samples were prepared in ultra-pure water spiked with active substances atQuantitative Analysis of Bioactive Copper Complexes 379 120, 150, and 180 μg mL−1 by following the same procedure as for calibra-tion standards.Visible Spectrophotometric MethodAqueous primary stock solutions of 1 mg mL−1 copper(II) complexes with pullulan or dextran were prepared. All measurements were made at room temperature. Calibration solutions were prepared by appropriate dilution of the primary stock solution with ultra-pure water to obtain concentrations in the range 4–40 μg mL−1 and 6–180 μg mL−1 for the copper(II) complexes with pullulan and dextran, respectively. Quality-control standard solutions were prepared in the range of the calibration plot at different concentra-tions, in triplicate.Method ValidationLinearityBoth methods were validated for linearity, sensitivity, precision and accu-racy in accordance with International Conference on Harmonization Q2(R1) guidelines for validation of analytical procedures [24, 25]. Six-point calibra-tion plots were generated with appropriate concentrations of calibration standard solutions for both visible spectrophotometric and GPC methods. For the visible spectrophotometric method the range was optimized at 4–40 μg mL−1 for the copper(II) complex with pullulan and 6–180 μg mL−1 for the copper(II) complex with dextran. For the GPC methods the calibration range was 18–180 μg mL−1 for both formulations. Linearity was evaluated by use of the least-squares regression method using unweighted data.Precision and AccuracyBoth precision and accuracy were determined by analysis of QC standards (in addition to calibration standards) prepared in triplicate at different con-centrations covering the entire linear range. Precision is the repeatability of an analytical method under normal operational conditions. Inter-day, intra-day, and inter-instrument variation were studied to determine the interme-diate precision of the proposed analytical methods. QC standards at differ-ent concentrations were prepared in triplicates three different times in a day to study intra-day variation. The same procedure was followed for three different days to study inter-day variation. One set of QC standards at dif-I. Savic et al. 380ferent concentrations was reanalyzed using another HPLC system and visi-ble spectrophotometer, by proposed methods, to study inter-instrument variation. The relative standard deviation (RSD, %) of the predicted concen-trations from the regression equation was taken as precision. Accuracy is the amount (%) of active substance recovered by assay from a known added amount. Results from nine analyses at three concentrations covering the specified range was determined [26]. The repeatability of the method was determined by assaying six sample solutions of the highest test concentra-tion (180 μg mL−1) for the GPC method and 40 and 180 μg mL−1, respec-tively, for the copper(II) complexes with pullulan and dextran for the visible spectrophotometric method.SpecificityThe specificity of method was assessed by comparing chromatograms (GPC) and scans (visible spectrophotometry) obtained from the drug and from a blank solution of the excipients in water without the drug. The ex-cipients chosen were those commonly used in tablet formulations, and in-cluded lactose, starch, silica, PVP-K30, and magnesium stearate. The drug to excipient ratio used was similar to that in formulations F1 and F2.LOD and LOQThe limits of detection (LOD) and quantification (LOQ) were evaluated by use of the equations LOD =3.3S0/b and LOQ =10S0/b, were S0 and b are the standard deviation and slope of the calibration plot [27–30].Robustness of the GPC MethodRobustness should show the reliability of an analysis in respect of deliber-ate variation of the method conditions. To determine method robustness, experimental conditions were deliberately altered. The flow rate of the mo-bile phase was 1 mL min−1. To study the effect of flow rate on the resolu-tion, it was changed by ±0.5 units to 0.5 and to 1.5 mL min−1 while the mo-bile-phase components were held constant in accordance with the method. The effect of column temperature on resolution was studied at 20 and 30°C instead of 25°C.Quantitative Analysis of Bioactive Copper Complexes 381 Robustness of Visible Spectrophotometric MethodFor evaluation of method robustness, the wavelength was changed by ±10 units from 630 to 650 nm, and the solvents (water and acetonitrile) and temperature were altered.Results and DiscussionGPC MethodA GPC method was developed which can be conveniently used for routine quality control analysis of the two active substances in pharmaceutical for-mulations. The chromatographic conditions were optimized to provide good assay performance. The mobile phase for each formulation was se-lected on the basis of its solubility. The copper(II) complexes with polysac-charides were quite soluble in water and acetonitrile. During optimization of the method three different columns were used: Zorbax PSM-60 (M w 500–10000 g mol−1), Zorbax PSM-300 (M w 3000–300 000 g mol−1) and Zorbax PSM Bimodal-S (M w 500–1 × 106 g mol−1) with acetonitrile–water combina-tions in different ratios as mobile phases. Chromatographic separation was achieved on the Zorbax PSM-60 column. Satisfactory separation of the stan-dards used was achieved with ultra-pure water as mobile phase. If acetoni-trile was used instead of water, resolution of the copper(II) complex was unsatisfactory. Maximum absorption of the copper(II) complexes was at 640 nm and this wavelength was chosen for the analysis. The retention times of the copper(II) complexes with pullulan and dextran were 5.820 and 5.619 min, respectively. The total run time was short for both drugs. The chromatograms are shown in Figs 2 and 3.T he chromatographic data column efficiency and peak asymmetry were measured for the active substances. The number of theoretical plates, N, was 161662 for Cu(II)–RLMP and 133130 for Cu(II)–RLMD, which indi-cates that column efficiency is satisfactory [HETP = 0.001 for Cu(II)–RLMP and 0.002 for Cu(II)–RLMD]. The peak asymmetry values of 0.67 for Cu(II)–RLMP and 0.69 for Cu(II)–RLMD indicate the peaks are not ideally symmet-ric, i.e. are not Gaussian.I. Savic et al. 382Fig. 2. Typical chromatograms showing elution of (a) the copper(II) complex with pullulan and (b) the copper(II) complex with dextran, at concentrations of 20 μg mL−1,with their respective retention timesT he methods were specific, because none of the excipients interfered with the active substances of interest. Hence, the methods were suitable for assay of the antihypocupremical formulations. Six-point calibration plots were constructed after analysis of calibration standards and found to be lin-ear (r≥ 0.997) for both active substances over their calibration ranges (Table I). The precision of the fit was further confirmed by the SE, at 95% confidence limits, of the intercepts and slopes. A one-way analysis of vari-ance (ANOVA) test [31, 32] was performed on the basis of the values ob-served for each pure drug during replicate analysis of standard solutions.Quantitative Analysis of Bioactive Copper Complexes 383 Calculated F-values (F calc) were found to be less than the critical F-value (F crit) at 5% significance levels (Table II). The GPC method was accurate, pre-cise, reproducible, and very sensitive. All the validation data for the two drugs were within specified limits (Table I). Accuracy and precision were determined by construction of calibration plots, two on the same day (intra-day) and a third on a different day (inter-day) and by use of different in-struments. Intra-day and inter-day precision (as RSD) at different concen-trations were <2%, indicative of the high precision of the GPC method. The calculated LOQ and LOD concentrations confirmed that the method was sufficiently sensitive. Hence, the method was suitable for assay of the anti-hypocupremical formulations.Fig. 3. Typical chromatograms showing elution of formulations F1 (a) and F2 (b), at concentrations of 20 μg mL−1, with their respective retention timesI. Savic et al.384Table I. Validation data for the GPC method of analysis of the complexes of copper(II)with pullulan and dextranValidationCu(II)–RLMP Cu(II)–RLMD dataRange18–180 18–180 (μg mL−1)Regressiony = 2.01184x + 3430.556 y = 4.62023x + 3217.09equationSE of slope 5.001 × 10−3 4.530 × 10−3SE of inter-6.246 × 10−3 5.340 × 10−4ceptCorrelation0.9976 0.9982 coefficient(r)Limit ofquantifica-1.46 1.59 tion(μg mL−1)Limit ofdetection0.48 0.53 (μg mL−1)Drug concentration (μg mL−1)Precision120 150 180 120 150 180150.94120.07179.26148.75Mean 119.12181.02RSD (%) 0.82 ± 0.49 1.32 ± 0.81 0.92 ± 0.620.75 ± 0.080.8 ± 0.19 1.5 ± 0.13Recovery99.26 99.16 100.56 100.58 100.62 99.58(%)Two calibration graphs were generated on the same day and on three consecutivedays (n = 3). The six standard concentrations were evenly distributed in the linear range.Precision and accuracy were determined by analysis of quality-control samples at three concentrations. Results show the precision of the method at three concentrations withinthe calibration range. The slopes are presented as mean ± SD with RSD (%) given in pa-renthesesW hen the chromatographic conditions flow rate and column tempera-ture were deliberately varied, no significant change in the assay value wasobserved. System suitability data, for example tailing and the RSD values,are well within accepted limits. Tailing was 1.0 and 0.9 and RSD was 0.8and 0.6% for flow rate and column temperature variations, which confirmsthe robustness of the method.Table II . Results from one-way ANOVA test of the linearity of assay of Cu(II) complexsolutions by the proposed methods F -value Source of varia-tion Degree of freedom(d.f.)Sum of squares (SS) Mean sum of squares (MS) F calc F critGPC method Between group 46.7750 × 10−3 1.6940 × 10−3 Within group 20 6.4223 3.2112 × 10−1Total 24 6.42910.0053 2.8661a Visible spectrophotometric methodBetween group4 8.4321 × 105 2.1080 × 105 Within group30 6.3665 × 1010 2.1222 × 109 Total 34 6.3666 × 1010 0.9999 2.6896ba Theoretical value of F (4, 20) based on one-way ANOVA test at the P = 0.05 level of significanceb Theoretical value of F (4, 30) based on one-way ANOVA test at the P = 0.05 level of significanceVisible Spectrophotometric MethodThe development of a simple, rapid, sensitive, and accurate analytical method for routine quantitative analysis of samples will reduce unneces-sary tedious sample preparation and the cost of materials and labor. Cop-per(II) complexes with the polysaccharides pullulan and dextran are visible light-absorbing molecules that absorb at a specific wavelength and this fact has been successfully used for their quantitative analysis by a visible spec-trophotometric method. The λmax of the copper(II) complexes in aqueous media was found to be 640 nm. The visible spectra of the active substances and their formulations are shown in Figs 4 and 5.T he correlation coefficients were highly significant (Table III ). Like the GPC method, a one-way ANOVA test [31, 32] was also performed for the visible spectrophotometric method based on the values observed during replicate measurement of standard solutions of the pure drug. F calc was found to be less than F crit at the 5% significance level for this method also (Table II ).Fig. 4. Visible absorption spectra of aqueous solutions of (а) complex Cu(II)–RLMP and(b) formulations F1All the method-validation data are well within the limits as specified inthe ICH Q2(R1) guidelines, as shown in Table III.(b) formulations F2Table III. Validation data for the visible spectrophotometric method of analysis ofcopper(II) complexes with pullulan or dextranValidation data Cu(II)–RLMP Cu(II)–RLMDRange (μg mL−1) 4–40 6–180Regressiony = 0.0020 x + 0.03185 y = 0.0001 x + 0.04117equationSE of slope 3.891×10−2 6.224×10−2SE of intercept 2.546×10−4 4.331×10−3Correlation0.9996 0.9989 coefficient (r)Limit ofquantification0.65 0.79 (μg mL−1)Limit of detection0.21 0.28 (μg mL−1)Drug concentration (μg mL−1)Precision12 15 18 120 150 180Mean 11.81 14.88 17.67 120.48 146.72 182.07RSD (%) 1.7±0.130.66±0.080.82±0.22 1.2±0.080.98±0.03 1.1±0.71Percent recovery 98.40 99.23 98.2 100.40 97.81 101.15 Two calibration graphs were generated on three consequent days (n = 3) with aminimum of six concentrations evenly distributed throughout the entire range. The accuracy, represented by percent recovery, and precision were determined using QC solutions. Precision (RSD, %) is calculated as mean ± SD with n=3 for each con-centrationF or evaluation of method robustness, the wavelength range and the temperature of analysis were changed. These changes had no effect on the absorbance of the complexes. RSD was 0.45 and 0.70% for variation of wavelength range and temperature, respectively. These changes thereforehad no significant effects on assay results, suggesting the method wasrobust.Applicability of the MethodsThe methods were used for analysis of copper(II) complexes in antihypocu-premical formulations. The results obtained by use of the methods were compared statistically using a point hypothesis test [33, 34]. For both drugsrecovery from the formulations was 93.16–101.87% when assayed by use ofthe GPC method and 99.61–100.32% when assayed by use of the visible spectrophotometric method (Table IV). Table IV shows that calculated F and t values at the 95% confidence level were less than the theoretical values, confirming no significant differences between the performance of the GPC and the visible spectrophotometric methods. The visible spectrophotometric method is, therefore, a cost-effective and time-saving alternative to the GPC method.Table IV. Analysis of the complexes by the GPC and visible spectrophotometric methodsRecovery (mean ± RSD; n = 3)Formulation Component Visiblespectrophotometricmethod GPC methodtvalue aFvalue aF1 Cu(II)–RLMP100.32 ± 1.71 93.16 ± 0.42 1.03F2Cu(II)–RLMD99.61 ± 1.45 101.87 ± 1.53 0.922.06a Theoretical F (ν1 = 4, ν2 = 4) and t (ν = 8) values at the 95% confidence level are 6.39and 2.306, respectivelyRSD, relative standard deviationQ uality-control tests were performed on formulations F1 and F2; the results are listed in Table V. Friability, disintegration, and hardness of the formulations were within the prescribed limits.Table V. Results from quality-control testing of formulations F1 and F2 in accordancewith the United States PharmacopeiaFormulation F1a F2aFriability 0.002 ± 3.6·10-40.0031 ± 1.2·10-4 Hardness (kPa) 70 ± 0.029 60 ± 0.018Weight variation (%) −0.95 to 2.1 (± 0.048) −1.37 to 2.26 (± 0.052)Disintegration Within 15 min (3.2-4.3 min)(± 0.10) Within 15 min (2.4-4.05)(± 0.08)a Uncoated tablet. Mean ± SD (n = 3 for checking hardness and disintegration; n = 10 for friability testing; and n = 20 for weight variation)ConclusionsThe purpose of this work was to validate the two analytical methods for quantitative analysis of copper(II) complexes with the polysaccharides pul-lulan and dextran in pharmaceuticals. The methods proved to be simple, precise, and accurate. 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