Uptake and transport of the ACE-inhibitor ceronapril (SQ 29852) by monolayers of human intestinal absorptive (Caco-2) cells in vitro

The angiotensin-converting enzyme (ACE)-inhibitor ceronapril (SQ 29852) is shown to be a substrate of the intestinal dipeptide transporter. Uptake by Caco-2 cells, grown as confluent monolayers, follows a major saturable pathway (K_m, 0.91 ± 0.11 mM; 90% at 1 mM) together with a minor passive component (k_J, 32.3 ± 6.6 ng (10⁶ cells)⁻¹ (20 min)⁻¹. Uptake was inhibited by competition with dipeptides such as l-AIa-l-Pro (K_i, 2.96 mM) and l-Phe-Gly (K_i, 3.84 mM) but not by cephalosporins such as cephalexin. In contrast, transport was non-saturable, flux increased linearly with concentration and data were consistent with a passive transepithelial transport mechanism. Transport profiles showed a biphasic dependence upon time with an initial flux of 0.83 ± 0.02 ng insert⁻¹ min⁻¹ (k₁) and a terminal value of 1.65 ± 0.08 ng insert⁻¹ min⁻¹ ((k₂) at 100 μM. It is concluded that the basolateral efflux is retarded so that the passive paracellular transport controls the overall transepithelial transport characteristics in the Caco-2 model. Carrier-mediated uptake into intestinal enterocytres, followed by rate-limiting basolateral efflux, may explain the extended t_max in vivo following oral administration.     

The influence of pH, temperature and buffers on the degradation kinetics of cefetamet pivoxil hydrochloride in aqueous solutions

The first-order hydrolysis kinetics of cefetamet pivoxil (CP) were investigated as a function of pH, temperature and buffers. The degradation was followed by HPLC. Buffer catalysis was observed in acetate and phosphate buffers. The pH–rate profiles for hydrolysis of cefetamet pivoxil were obtained at 333, 343, 353 and 363 K. The pH–rate expression was k_pH=k_H⁺a_H⁺+k_H2Ok_OH⁻a_OH⁻, where k_H⁺ and k_OH⁻ are the second-order rate constants (mol⁻¹ l s⁻¹) for hydrogen ion activity and for hydroxyl ion activity respectively, and k_H2O is the pseudo-first-order rate constant (s⁻¹) for spontaneous reaction under the influence of water. The pH–rate profile was characteristically U-shaped. Maximum stability was observed in the pH region from 3 to 5.     

Effect of pharmaceutical excipients on aqueous stability of rabeprazole sodium

The chemical stability of a proton-pump inhibitor, rabeprazole sodium, was evaluated in simulated intestinal fluid (pH 6.8) containing various ‘Generally Recognized As Safe (GRAS)’-listed excipients, including Brij^® 58, Poloxamer 188, Cremophor RH40, Gelucire 44/14 and PEG 6000. After incubation at 37 and 60 °C, the amounts of rabeprazole and its degradation product, thioether-rabeprazole, were quantitated by HPLC analysis. The main degradation product was separated and characterized by LC/MS. The degradation of rabeprazole followed first-order kinetics. In the absence of any excipients, the rate constants (k) obtained at 37 and 60 °C were 0.75 and 2.78 h⁻¹, respectively. In contrast, the addition of excipients improved its stability. Among several excipients tested in this study, Brij^® 58 displayed the greatest stabilizing effect. For instance, at 37 and 60 °C, Brij^® 58 reduced the k values to 0.22 and 0.53 h⁻¹, respectively. The stabilizing mechanisms of these hydrophilic polymeric excipients with optimal HLB values could be partially explained in terms of their solubilizing efficiency and micellar formation for thioether-rabeprazole. In conclusion, rabeprazole formulations that contain suitable excipients would improve its stability in the intestinal tract, thereby maximizing bioavailability.     

Disposition kinetics and urinary excretion of levofloxacin on concomitant administration with meloxicam in cross-bred calves

The disposition kinetics and urinary excretion study of levofloxacin was conducted in 5 male cross-bred calves following its single intravenous administration (4 mg kg⁻¹) concurrently with meloxicam (0.5 mg kg⁻¹). Levofloxacin was estimated by microbiological assay. The drug levels above MIC₉₀ in plasma, were detected up to 10 h. Disposition kinetic parameters were calculated by two-compartment open model. Rapid distribution of levofloxacin was evidenced by a small distribution half-life (0.13 ± 0.01 h) and high K₁₂/K₂₁ ratio (2.21 ± 0.15). High ratio of AUC/MIC (90.2 ± 3.41) indicated good antibacterial activity of levofloxacin. The AUC, Vd_area, elimination half-life, MRT and total body clearance were 9.02 ± 0.34 μg ml⁻¹ h, 1.38 ± 0.05 l kg⁻1, 2.16 ± 0.08 h, 2.58 ± 0.11 h and 0.45 ± 0.02 l kg⁻¹ h⁻¹, respectively. About 38.4% of the administered dose of levofloxacin was excreted in urine within 24 h. A suitable intravenous dosage regimen for levofloxacin would be 1.8 mg kg⁻¹ repeated at 8 h intervals when prescribed with meloxicam in calves.     

Effect of temperature on limitation by MK-801 of firing of action potentials by spinal cord neurons in cell culture

The anticonvulsant, MK-801, limited sustained high frequency repetitive firing of sodium-dependent action potentials by mouse spinal cord neurons in monolayer dissociated cell culture. Limitation was voltage- and temperature-dependent and was accompanied by decreasing rate of rise of action potentials until firing ceased during the 400 ms depolarizations. The IC₅₀ for limitation was 2 × 10⁻⁷ M at 37°C, 6.4 × 10⁻⁷ M at 35°C, and 4 × 10⁻⁵ M at 23°C. The relationship between the percentage of neurons capable of sustained repetitive firing and MK-801 concentration at 33°C was biphasic. The first phase (about 50%) of limitation had IC_50a = 1.5 × 10⁻⁷ M, and the second had IC_50b = 2 × 10⁻⁴ M; the midpoint of the connecting plateau was 10⁻⁵ M. At temperatures below 37°C, the current needed to achieve maximal firing increased. The maximal rate of rise, maximal firing frequency and sensitivity to MK-801 of action potentials elicited by 1 ms stimuli decreased at temperatures below 37°C. Passive membrane properties were unchanged. Slow firing and a temperature-sensitive conformational change in voltage-activated sodium channels could account for the higher concentrations of MK-801 required to block sodium-dependent action potentials at temperatures below 37°C.     

In vitro studies with ICI 169,369, a chemically novel 5-HT antagonist

ICI 169,369 is a chemically novel 5-HT antagonist that has higher affinity for the 5-HT₂ binding sites in rat cortex than it has for 5-HT₁ sites (K_i 1.79 × 10⁻⁸ and 1.58 × 10⁻⁶ M, respectively). In isolated tissue preparations ICI 169,369 was shown to be a competitive antagonist of 5-HT on the rabbit aorta, pig coronary artery and rat caudal artery. In the latter preparation it had a similar pA₂ value to ketanserin (pA₂ 8.18 ± 0.5 and 8.42 ± 0.06, respectively). Unlike ketanserin, which was inactive, ICI 169,369 was a non-surmountable antagonist at the rat stomach fundus 5-HT ‘D’ receptor, recently reclassified as 5-HT_IC. It was inactive ( > 10⁻⁶ M) at the 5-HT₃ receptors found in the isolated perfused rabbit heart and the myenteric plexus of the guinea-pig ileum. At receptors other than those for 5-HT (₁, ₂, β₁, β₂, H₁, H₂ and muscarinic), ICI 169,369 was inactive at concentrations of either 10⁻⁶ or 10⁻⁵ M. Thus the profile of ICI 169,369 should make it useful in the analysis of the role of 5-HT in physiological and pathological states.     

Degradation kinetics of vincristine sulphate and vindesine sulphate in aqueous solutions

The degradation kinetics of the antineoplastic drugs, vincristine and vindesine, have been studied in the pH range from − 2 up to 11 at 80°C. A stability-indicating HPLC system with UV detection was utilized for the analysis of vincristine and vindesine in the reaction solutions. The influences of external factors (e.g. pH, buffer concentrations, ionic strength and temperature) on the degradation rate have been studied systematically. The relationship between pH and log k_obs was modelled by using a non-linear least-squares curve-fitting computer program. From this plot thepK_a values of vindesine have been calculated. This plot also showed that vincristine was most stable at pH 4.8 and vindesine at pH 1.9.     

Stability of flavone-8-acetic acid (LM975) in aqueous solutions by high-performance liquid chromatography

A high-performance liquid chromatographic method has been developed to investigate the stability of solutions of flavone-8-acetic acid (LM975) during preparation and storage. LM975 (20 μg ml⁻¹ in PBS) was found to be completely stable for 10 days at 80°C as long as light was rigorously excluded. The drug showed no significant adsorption to containers of different materials or to two filtration units tested. Drug degradation did occur however, on exposure to light. In normal laboratory light the t_0.95 (5% degraded) was 30.3 min, in intense natural light (laboratory window sill) the t_0.95 was 3.3 min and in intense artificial light (100 W bulb at 10 cm) the t_0.95 was 13.8 min. NMR and mass spectral analysis of the isolated degradation product implied the formation of the decarboxylated product, 8-methyl flavone. It is suggested that care be taken to exclude light during the preparation, storage and infusion of solutions of flavone-8-acetic acid.     

Alkaline hydrolysis of methyl, ethyl and n-propyl 4-hydroxybenzoate esters in the liquid and frozen states

The alkaline hydrolysis of the methyl, ethyl and n-propyl esters of 4-hydroxybenzoic acid was studied in the liquid and frozen states in sodium hydroxide solutions. The temperature range was −26 to 60°C. Significant acceleration of the reaction rate was evident in the frozen state compared with rates found at liquid state temperatures. The maximum reaction rate in the frozen state occurred in the temperature range −12 to −10°C. Methyl 4-hydroxybenzoate showed more than a 20-fold increased rate constant from 7.17 × 10⁻⁶ s⁻¹ at 30°C to 1.53 × 10⁻⁴ s⁻¹ at −9°C in a 1.00 × 10⁻² M solution of sodium hydroxide. Rate constants in the liquid and frozen states followed pseudo first-order kinetics over 2–4 half-lives of reaction. Data were fitted to a theoretical model describing the reaction rate in the frozen state as dependent upon the increased concentration of solutes in liquid vesicles in the frozen state and the predicted reduction in the reaction rate constant with temperature decrease. Although the data exhibited similar trends to that predicted by the model, there was frequently a 50% difference in the rates observed compared with those predicted. This study has clearly demonstrated that there is a significantly increased rate of hydrolysis of these esters in the frozen state. This is a further indication that it cannot be assumed that drugs stored in solution will necessarily be stabilized, or their stability enhanced, on freezing. Storage under refrigeration conditions (4–8°C) results in enhanced shelf-lives compared with deep-freeze storage at −20°C under the conditions of this study.     

Investigation of penbutolol—iron(III) complex and its spectrophotometric determination in tablets

It has been established that penbutolol reacts with iron(III) chloride in the presence of ammonium thiocyanate to form a pink complex (2:1) that is soluble in chloroform with a maximum absorbance at 478 nm. By application of the methods of Sommer and Job involving non-equimolar solutions, the conditional stability constant (log k′) of the complex at the optimum pH of 1.5±0.02 and an ionic strength of (μ) 0.14 M, was found to be 5.769. The molar absorptivity at 478 nm was 136 l mol⁻¹ cm⁻¹ at pH 1.5±0.02. The validity of Beer's law has been tested in the concentration range 3–18 × 10⁻⁴ M; the relative standard deviation (n = 8) was 1.52–3.21%. The proposed method was found to be suitable for the accurate, simple and rapid analysis of penbutolol in the bulk drug and in tablets.     

Microcalorimetric investigation of the toxic action of ammonium ferric(III) sulfate on the metabolic activity of pure microbes

A series of calorimetric experiments were performed to investigate toxic action of ammonium ferric sulfate (AFS) on Bacillus subtilis, Pseudomonas putida and Candida humicola. The power–time curves of micro-organism metabolism were obtained, and the action of them by addition of AFS was studied. C. humicola, B. subtilis and P. putida were inhibited completely when the concentrations were up to 320.0, 160.0 and 160.0 μg mL⁻¹, respectively. The relationships between growth rate constant (k) and doses of AFS were approximately linear for three microbes, P. putida for 10.0–160.0 μg mL⁻¹ (R = −0.9746), B. subtilis for 0–160.0 μg mL⁻¹ (R = −0.9868) and C. humicola for 10.0–320.0 μg mL⁻¹ (R = −0.9955). The total heat dissipated per milliliter (Q_T) for three microbes remained balance approximately during the lower doses, P. putida and B. subtilis less than the dose of 20.0 μg mL⁻¹, 0.56 ± 0.01 and 0.26 ± 0.01 J mL⁻¹, respectively, C. humicola less than the dose of 40.0 μg mL⁻¹, 0.58 ± 0.03 J mL⁻¹. The biomass and OD₆₀₀ of three micro-organisms growth in the absence of AFS also were obtained. The power–time curve of C. humicola growth coincided with its turbidity curve. It elucidates that microcalorimetric method agreed with the routine microbiology method.     

Inhibitory effects of cilnidipine on peripheral and brain N-type Ca2+ channels expressed in BHK cells

We investigated differences in electrophysiological characteristics between peripheral and central N-type Ca²⁺ channels, containing _1B-a and a_1B-c, respectively. In addition, we examined the inhibitory effects of cilnidipine, a dihydropyridine (DHP) derivative, on both channels. Both _1B subunits were transiently expressed in BHK cells, and then analyzed using the whole-cell patch-clamp technique. The current–voltage relationship showed that _1B-c currents were activated at more negative potentials than _1B-a currents. The voltage-dependent steady-state inactivation and activation showed that the V_1/2 values for inactivation and activation of _1B-c (−88.5±1.3 and −33.2±1.3 mV) were both significantly more negative than those for _1B-a (−83.3±1.3 and −27.9±2.3 mV). Despite the different electrophysiological characteristics of these two N-type channels, cilnidipine blocked both with similar potency within the range 0.1 to 10 μM. Furthermore, cilnidipine had no effect on the I–V relationships or the steady-state inactivation curves. Our data indicate that the spliced positions of _1B-a and a_1B-c may affect not only their voltage-sensing abilities but also the kinetics of channel activation and inactivation. The data also suggest that cilnidipine binds to sites independent of those controlling voltage-sensing and channel kinetics in these _1B subunits.     

Comparison of effects of formoterol and BRL 37344 on isolated term-pregnant rat myometrial strips in vitro

This study was designed to compare the effects of β-adrenoceptor agonists formoterol and BRL 37344 on spontaneous contractions and the levels of cAMP and cGMP of myometrial strips isolated from timed-pregnant rats. Myometrial strips were obtained from term-pregnant Wistar albino rats (n = 12), mounted in organ baths and tested for changes in isometric tension in response to formoterol and BRL 37344. We evaluated the effect of increasing concentrations of formoterol and BRL 37344 on oxytocin-induced myometrial contractions and on contractions of myometrial smooth muscle pretreated with metoprolol, ICI 118.551 and SR 59230A (β₁, β₂, β₃-adrenoceptor antagonist, respectively, 10^− 6 M). Effects of formoterol and BRL 37344 on cAMP and cGMP levels in isolated myometrial strips (n = 6) were evaluated by radioimmunoassay kits. Formoterol (10^− 12–10^− 8 M) and BRL 37344 (10^− 11–10^− 5 M) concentration-dependently decreased the amplitude of oxytocin-induced contractions. E_max value (100%) of formoterol was increased significantly more than E_max value (70.6%) of BRL 37344 (P < 0.05), with no change in pD₂ value (9.54 ± 0.12 and 9.12 ± 0.12, respectively). The inhibition of the amplitude of oxytocin-induced contractions by formoterol was antagonized with ICI 118.551 (10^− 6 M), but they were not changed by metoprolol (10^− 6 M) or SR 59230A (10^− 6 M). The inhibition of the amplitude of oxytocin-induced contractions by BRL 37344 were antagonized with SR 59230A (10^− 6 M), but they were not changed by metoprolol (10^− 6 M) or ICI 118.551 (10^− 6 M). Formoterol and BRL 37344 increased cAMP levels. BRL 37344 increased cGMP levels in BRL 37344 group more than control group, but this increase is less significant than cAMP levels (P > 0.05). Formoterol and BRL 37344 decreased amplitude of myometrial contractions with similar potency, but efficacy of formoterol was better than BRL 37344.     

LC/ESI-MS method for the determination of trimetazidine in human plasma: application to a bioequivalence study on Chinese volunteers

A rapid liquid chromatography electrospray ionization mass spectrometry (LC/ESI-MS) method with good sensitivity and specificity has been developed and validated for the identification and quantification of trimetazidine in human plasma. Trimetazidine and lidocaine (internal standard) were isolated from plasma samples by protein precipitation with methanol. The chromatographic separation was accomplished on a Xterra MS C₁₈ Column (150 mm × 4.6 mm, 5 μm particle size) with the mobile phase consisting of methanol and water (40:60, v/v) (pH 2.0, adjusted with trifluoroacetic acid), and the flow rate was set at 0.6 mL/min. Detection was performed on a single quadruple mass spectrometer by selected ion monitoring (SIM) mode (m/z 267.0 for trimetazidine and m/z 235.0 for lidocaine) with the retention time at about 3.47 and 5.05 min, respectively. The calibration curve for trimetazidine was satisfactory with regression coefficient 0.9995 over the range of 2.5–100 ng/mL in the plasma. The LOQ (S/N = 10) was accordingly 2.5 ng/mL. The intra-day and inter-day precision expressed as relative standard deviation was 2.83–6.10% and 4.83–5.82%. The method was successfully applied to investigate the bioequivalence between two kinds of tablets (test versus reference product) in 19 healthy male Chinese volunteers. After a single 20 mg dose for the test and reference product, the resulting mean of major pharmacokinetic parameters such as AUC_0–24, AUC_0−∞, C_max, T_max and t_1/2 of trimetazidine were (673.1 ± 117.6 ng h mL⁻¹ versus 652.3 ± 121.9 ng h mL⁻¹), (717.1 ± 120.9 ng h mL⁻¹ versus 692 ± 128.6 ng h mL⁻¹), (74.85 ± 12.13 ng mL⁻¹ versus 71.93 ± 14.32 ng mL⁻¹), (2.312 ± 0.663 h versus 2.211 ± 0.608 h) and (4.785 ± 0.919 h versus 4.740 ± 0.823 h), respectively, indicating that these two kinds of tablets were bioequivalent in the Chinese population.     

The pharmacokinetics of pulmonary insulin in the in vitro isolated perfused rat lung: Implications of metabolism and regional deposition

The pharmacokinetics of several lung disposition pathways for pulmonary insulin were studied and modeled in the isolated perfused rat lung (IPRL). Insulin solution was administered by forced instillation into the airways of the IPRL as 0.1 or 0.02 ml doses of coarse spray, with or without bacitracin (BAC), N-ethylmaleimide (NEM) and atrial natriuretic peptide (ANP). Each insulin absorption profile was fitted to a kinetic model that incorporated the distribution fraction of the dose reaching the lobar region (DF) and the rate constants for absorption into perfusate (k_a) and non-absorptive loss (k_nal); k_nal was shown to be due to the sum of mucociliary clearance and metabolism. Insulin absorption occurred largely by passive diffusion with values for k_a = 0.39–0.50 h⁻¹. With DF = 0.91 following 0.1 ml doses, 11.9 ± 3.4% of bioavailabilities were observed in 1 h. In contrast, derived values for k_nal = 2.34–3.45 h⁻¹ were significantly larger than the rate constant for mucociliary clearance determined previously in this IPRL (0.96–1.74 h⁻¹) due to lung metabolism. Indeed, BAC, but neither NEM nor ANP, was found to decrease the value of k_nal, which suggested that BAC-inhibitable lung ectopeptidases, and not insulin degrading enzyme (IDE), were responsible for this pulmonary metabolism. Shallower lung distribution with DF = 0.73 following 0.02 ml doses resulted in reduced values for k_a = 0.27 h⁻¹ and k_nal = 2.79 h⁻¹, indicating that these kinetic processes may be lung-region dependent, even within this model and emphasizing the likely importance of reliable lung deposition in vivo.     

Sodium pump activity and contraction of renal artery from spontaneously hypertensive rats

We investigated the hypertensive changes in renal arteries isolated from 21-week-old spontaneously hypertensive rats (SHR), and from age-matched normotensive Wistar-Kyoto rats (WKY). The maximam contraction of renal arteries from SHR in response to norepinephrine (NE), serotonin (5-HT) and KCl was greater than that of arteries from WKY. The threshold and EC₅₀ concentrations of NE, 5-HT and KCl were not significantly different between SHR and WKY. Contraction induced by removal of K⁺ was inhibited by 10⁻⁸ M prazosin. Less than 10⁻⁷ M NE in K⁺-free solution did not cause contraction. Addition of 5.9 mM KCl to K⁺-free solution in the presence of 10⁻⁵ M NE induced relaxation, which was followed by contraction to about the same level as that before KCl addition. The duration of the K⁺-induced relaxation in SHR (22.4 ± 0.9 min) was slightly, but significantly shorter than that in WKY (26.6 ± 0.8 min) arteries. In K⁺-free solution with reduced Na⁺, the duration of the relaxation induced by KC1 was shorter than that in the normal solution, for both SHR (13.8 ± 0.3 min) and WKY (14.1 ± 0.5 min). Such differences could be caused by increased influx and decreased efflux of Ca²⁺, which depend on the Na⁺ concentration and are related to the Na⁺-Ca²⁺ exchange. The results suggest that enhanced renal vascular reactivity in hypertension may depend on structural changes and increased Na⁺ pump activity.     

A highly sensitive and selective method for the determination of Leukotriene B4 in human plasma by negative ion chemical ionization/gas chromatography/tandem mass spectrometry

We have developed a highly sensitive and highly selective method for the determination of Leukotriene B₄ (LTB₄) in human plasma using negative ion chemical ionization/gas chromatography/tandem mass spectrometry (NICI/GS/MS/MS) analysis. The developed method was summarized as follows. Deuterated LTB₄ (d₄-LTB₄) was added to human plasma samples as an internal standard, and samples were extracted by a Sep-pak C18 column. Extracted LTB₄ was derivatized into the pentafluorobenzyl ester of bis-trimethylsilyl ether (PFB-TMS-LTB₄) and quantified on the basis of selected reaction monitoring (SRM) at m/z 299 of [M-PFB-2TMSOH]⁻ by NICI/GC/MS/MS analysis, which was the product ion of [M-PFB]⁻. The detection limit for the quantification of LTB₄ in human plasma was 10 pg ml⁻¹, sufficiently sensitive to determine the concentrations of endogenous LTB₄ in human plasma. The plasma level of LTB₄ measured in healthy male volunteers was 33.85 ± 33.91 pg ml⁻¹ (mean ± S.D. in six volunteers). The technique of MS/MS used in this method offers much greater sensitivity and selectivity than single-stage mass spectrometry. The developed method showed good reproducibility with a simple and rapid extraction procedure, and would be useful for examining the relationship between various disease states and the levels of LTB₄ in biological fluids.     

Rheological properties of three component creams containing sorbitan monoesters as surfactants

The objectives of the present study were (1) to evaluate the effect of formulation ingredients on the release rate of Ubiquinone from its adsorbing solid compact; and (2) to prepare and evaluate an optimized self-nanoemulsified tablet formulation. A three factor, three-level Box–Behnken design was used for the optimization procedure, with the amounts of copolyvidone (X₁), maltodextrin (X₂) and microcrystalline cellulose (X₃) as the independent variables. The response variable was cumulative percent of Ubiquinone emulsified in 45 min with constraints on weight, flowability index, tensile strength, friability and disintegration time of the dry powdered emulsion and the resultant compact. Based on the experimental design, different Ubiquinone release rates and profiles were obtained. Mathematical equations and response surface plots were used to relate the dependent and independent variables. The regression equation generated for the cumulative percent emulsified in 45 min was Y₆=64.10−12.32X₁−4.36X₂−25.53X₃+6.99X₁X₂+3.97X₁X₃+9.70X₂X₃−8.98X₁²−16.22X₂²+17.10X₃². The optimization model predicted an 85.4% release with X₁, X₂ and X₃ levels of 66.6, 560.1 and 100, respectively. A new formulation was prepared according to these levels. The observed responses were in close agreement with the predicted values of the optimized formulation.     

Voltammetric determination of vitamins in a pharmaceutical formulation

Direct current polarography and differential pulse polarographic methods have been developed for the qualitative as well as quantitative analysis of vitamin B₁, B₂ and B₆. Thiamin (Vitamin B₁) produced a well-defined wave in 0.1 M KCl at pH 5.2 with E_1/2=−1.2 V and E_p=−1.22 V versus saturated calomal electrode (SCE). Riboflavin (Vitamin B₂) gave two distinct waves in Britton Robinson buffer at pH 1.8 with E_1/2 VALUES=−0.13 and −0.34 V versus SCE and at pH 6.5 with E_1/2=−1.10 V and E_p=−1.2 V versus SCE. Pyridoxin (Vitamin B₆) produced a well-defined wave in Britton Robinson buffer at pH 6.5 with E_1/2=−1.7 V and E_p=−1.68 V versus SCE. All the three Vitamins under study are reversibly reduced at the electrode surface. The number of electrons involved in the electrode process for vitamin B₁ and B₆ is one in each case where as for the two waves of B₂ it is one and two, respectively. This has been confirmed by the measurement of E_3/4–E_1/4 values and also from the log plot slopes for the reduction waves. The wave height of polarogram was found to be proportional to the vitamin concentration. The developed methods have been standardised for the determination of these compounds in pharmaceutical formulation. The concentration of Vitamin B₁, B₂ and B₆ are found to be 9.96, 9.92 and 3.01 mg, respectively in 240 mg of capsule powder of a standard company (name has not been disclosed due to secrecy purpose). The results have been found to be in excellent agreement to that claimed by the manufacturer. The observed data has been subjected to statistical analysis, which revealed high reliability and precision.     

Quantitative analysis of demeclocycline by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method suitable for the quality control of demeclocycline is described. The stationary phase is a poly(styrene-divinylbenzene) copolymer, kept at 60°C. The mobile phase comprises 2-methyl-2-propanol−0.2 M potassium phosphate buffer (pH 9.0)−0.02 M tetrabutylammonium hydrogen sulphate (pH 9.0)−0.01 M sodium edetate (pH 9.0)—water (8:10:15:10:57, m/v/v/v/v). The flow rate is 1 ml min⁻¹ and detection is performed at 254 nm. Official standards are compared and results for the analysis of a number of commercial bulk samples and preparations are presented. 4-Epidemeclocycline and demethyltetracycline are the main impurities. 4-Epidemethyltetracycline and 2-acetyl-2-decarboxamido-demeclocycline can also be present.