Towards a real-time release of blends and tablets using NIR and Raman spectroscopy at commercial scales.

Near Infrared and Raman spectroscopy-based Process Analytical Technology tools were used for monitoring blend uniformity (BU) and content uniformity (CU) for solid oral formulations. A quantitative Partial Least Square model was developed to monitor BU as real-time release testing at a commercial scale. The model having the R2, and root mean square error of 0.9724 and 2.2047, respectively can predict the target concentration of 100% with a 95% confidence interval of 101.85-102.68% even after one year. The tablets from the same blends were investigated for CU using NIR and Raman techniques both in reflection and transmission mode. Raman reflection technique was found to be the best and the PLS model was developed using tablets compressed at different concentrations, hardness, and speed. The model with R2 and RMSE of 0.9766 and 1.9259, respectively was used for the quantification of CU. Both the BU and CU models were validated for accuracy, precision, specificity, linearity, and robustness. The accuracy was proved against the HPLC method with a relative standard deviation of less than 3%. The equivalency for BU by NIR and CU by Raman was evaluated using Schuirmann's Two One-sided tests and found equivalent to HPLC within a 2% acceptable limit.

Development of Inline Near-Infrared Spectroscopy Method for Real-Time Monitoring of Blend Uniformity of Direct Compression and Granulation-Based Products at Commercial Scales.

Blending is a critical intermediate unit operation for all solid oral formulations. For blend uniformity testing, API content in the blend must be quantified precisely. A detailed study was conducted to demonstrate the suitability of inline NIR (near-infrared) spectroscopy for blend uniformity testing of two solid oral formulations: existing direct compression (DC) product with a multistep blending process and granulation-based product with API granules. Both qualitative and quantitative methods were developed at a laboratory scale using statistical moving block standard deviation (MBSD) and multivariate data analysis such as principal component analysis (PCA) and partial least squares (PLS) regression. The qualitative MBSD method demonstrated that there was no need for multiple steps for the existing DC product. Hence, a simplified single-step process was developed for blending. Quantitative PLS models for blending processes of both the products were developed, validated, and successfully implemented at a commercial scale for the real-time release of blends. Results obtained from the validated model were in good agreement with the current method of sampling and chromatography.

Optimization of a liquid chromatography method for the analysis of related substances in daclatasvir tablets using design of experiments integrated with the steepest ascent method and Monte Carlo simulation.

Analytical method for the determination of related substances (RS) in Daclatasvir tablets was optimised using quality by design (QbD) approach. Seven degradants (each more than 1.0%) generated during oxidation study, adversely affected the selectivity of the method. Coelution of the degradant peaks with API and known impurities, suggested failure in developing a stability indicating method. To overcome the shortcomings and develop a robust method, QbD principles were incorporated. Resolution was the critical quality attribute (CQA) and buffer pH, column oven temperature, gradient slope and flow rate were the critical method variables (CMVs) studied through design of experiments (DoE). Discovery of an unknown impurity (named as impurity D, about1.0%) was a key finding from this DoE study. The most crucial responses viz. Resolution between impurity D and the main peak and resolution between the main peak and impurity E demanded contradictory pH requirements. To select the right pH, responses were prioritised and eventually to attain the desired resolution between Daclatasvir and impurity E the value for pH was fixed to 3.0. Next, to improve resolution between impurity D and Daclatasvir, method of steepest ascent was applied to locate an apt value for column oven temperature. Accordingly, experiments were performed at different temperatures along the path of rapid increase in response. Finally, at 45 °C (pH :3.0), both the critical pairs were well resolved. The global optimum was determined through a Response surface methodology (RSM) design with pH and column oven temperature as critical factors. pH 3.0, column oven temperature 44 °C, % MP. B 45% and flow rate 1.0 mL min-1 was found to be the optimum condition. Further, the design space was complimented by establishment of a robust zone through Monte Carlo simulation and capability analysis. An analytical control strategy (ACS) was set up to ensure that the method repeatedly meets its acceptance criteria. The optimised method was successfully validated within the factor ranges mentioned in the ACS. Despite various intricacies, the QbD approach facilitated systematic optimisation of a stability indicating robust method.

Application of Quality by Design: Development to Manufacturing of Diclofenac Sodium Topical Gel.

The objective of the present study was to develop and optimize generic topical gel formulation of diclofenac sodium through quality by design approaches. The quality target product profile was set for the critical quality attributes of the gel. The key material variables like hydrophilic gelling agent carbopol and penetration enhancer kolliphor were optimized using design of experiments. A central composite design was used considering viscosity and cumulative percent diffusion of the drug after 0.5, 1, 2, 4 and 6 h as responses. The p values for all models generated for different responses were statistically significant (<0.5). Design space was established and verified at the laboratory scale. The predicted and observed values were in close agreement. The robustness of the formula was tested at a higher scale (10X and 200X). The capability index was calculated followed by Monte Carlo simulation and the Cpk values for all the responses were more than 1.33.

Fate of over 480 million inhabitants living in arsenic and fluoride endemic Indian districts: Magnitude, health, socio-economic effects and mitigation approaches.

During our last 27 years of field survey in India, we have studied the magnitude of groundwater arsenic and fluoride contamination and its resulting health effects from numerous states. India is the worst groundwater fluoride and arsenic affected country in the world. Fluoride results the most prevalent groundwater related diseases in India. Out of a total 29 states in India, groundwater of 20 states is fluoride affected. Total population of fluoride endemic 201 districts of India is 411 million (40% of Indian population) and more than 66 million people are estimated to be suffering from fluorosis including 6 million children below 14 years of age. Fluoride may cause a crippling disease. In 6 states of the Ganga-Brahmaputra Plain (GB-Plain), 70.4 million people are potentially at risk from groundwater arsenic toxicity. Three additional states in the non GB-Plain are mildly arsenic affected. For arsenic with substantial cumulative exposure can aggravate the risk of cancers along with various other diseases. Clinical effects of fluoride includes abnormal tooth enamel in children; adults had joint pain and deformity of the limbs, spine etc. The affected population chronically exposed to arsenic and fluoride from groundwater is in danger and there is no available medicine for those suffering from the toxicity. Arsenic and fluoride safe water and nutritious food are suggested to prevent further aggravation of toxicity. The World Health Organization (WHO) points out that social problems arising from arsenic and fluoride toxicity eventually create pressure on the economy of the affected areas. In arsenic and fluoride affected areas in India, crisis is not always having too little safe water to satisfy our need, it is the crisis of managing the water.

A simultaneous determination of related substances by high performance liquid chromatography in a drug product using quality by design approach.

The combination of Abacavir, Lamivudine and Dolutegravir is an anti-retroviral formulation that displays high efficacy and superiority in comparison to other anti-retroviral combinations. Analysis of related substances in this combination drug product was very challenging due to the presence of nearly thirty peaks including the three active pharmaceutical ingredients (APIs), eleven known impurities and other pharmaceutical excipients. Objective of this study was to develop a single, selective, and robust high performance liquid chromatography method for the efficient separation of all peaks. Initially, one-factor-at-a-time (OFAT) approach was adopted to develop the method. But, it could not resolve all the critical peaks in such complex matrix. This led to the advent of two different HPLC methods for the determination of related substances, one for Abacavir and Lamivudine and the other for Dolutegravir. But, since analysis of a single sample using two methods instead of one is time and resource consuming and thus expensive, an attempt was made to develop a single and robust method by adopting quality by design (QbD) principles. Design of Experiments (DoE) was applied as a tool to achieve the optimum conditions through Response surface methodology with three method variables, pH, temperature, and mobile phase composition. As the study progressed, it was discovered that establishment of the design space was not viable due to the completely distant pH requirements of the two responses, i.e. (i) retention time for Lamivudine carboxylic acid and (ii) resolution between Abacavir impurity B and unknown impurity. Eventually, neglecting one of these two responses each time, two distinguished design spaces have been established and verified. Edge of failures at both design spaces indicate high probability of failure. It therefore, becomes very important to identify the most robust zone or normal operating range (NOR) within the design space with low risk of failure and high quality assurance. For NOR establishment, Monte Carlo simulation was performed on the basis of which process capability index (Cpk) was derived. Finally, the selectivity issue problem faced due to the pH dependency and the dissimilar pH needs of the two critical responses was resolved by introducing pH gradient into the program. This new ternary gradient program has provided a single robust method. Thus, two HPLC methods for the analysis of the combination drug product have been replaced with a selective, robust, and cost effective single method.

Comparative study of arsenic removal by iron using electrocoagulation and chemical coagulation.

This research studied As(III) and As(V) removal during electrocoagulation (EC) in comparison with FeCl(3) chemical coagulation (CC). The study also attempted to verify chlorine production and the reported oxidation of As(III) during EC. Results showed that As(V) removal during batch EC was erratic at pH 6.5 and the removal was higher-than-expected based on the generation of ferrous iron (Fe(2+)) during EC. As(V) removal by batch EC was equal to or better than CC at pH 7.5 and 8.5, however soluble Fe(2+) was observed in the 0.2-µm membrane filtrate at pH 7.5 (10-45%), and is a cause for concern. Continuous steady-state operation of the EC unit confirmed the deleterious presence of soluble Fe(2+) in the treated water. The higher-than-expected As(V) removals during batch mode were presumed due to As(V) adsorption onto the iron rod oxyhydroxides surfaces prior to the attainment of steady-state operation. As(V) removal increased with decreasing pH during both CC and EC, however EC at pH 6.5 was anomalous because of erratic Fe(2+) oxidation. The best adsorption capacity was observed with CC at pH 6.5, while lower but similar adsorption capacities were observed at pH 7.5 and 8.5 with CC and EC. A comparison of As(III) adsorption showed better removals during EC compared with CC possibly due to a temporary pH increase during EC. In contrast to literature reports, As(III) oxidation was not observed during EC, and As(III) adsorption onto iron hydroxides during EC was only 5-30% that of As(V) adsorption. Also in contrast to literature, significant Cl(2) was not generated during EC, in fact, the rods actually produced a significant chlorine demand due to reduced iron oxides on the rod. Although Cl(2) generation and As(III) oxidation are possible using a graphite anode, a combination of graphite and iron rods in the same EC unit did not produce As(III) oxidation. However, a two-stage process (graphite anode followed by iron anode in separate chambers) was effective in As(III) oxidation and removal. The competing ions, silica and phosphate interfered with As(V) adsorption during both CC and EC. However, the degree of interference depends on the concentration and presence of other competing ions. In particular, the presence of silica lowered the effect of phosphate with increasing pH due to silica's own significant effect at high pHs.

Ferrous and ferric ion generation during iron electrocoagulation.

Our research on arsenate removal by iron electrocoagulation (EC) produced highly variable results, which appeared to be due to Fe2+ generation without subsequent oxidation to Fe3+. Because the environmental technology literature is contradictory with regard to the generation of ferric or ferrous ions during EC, the objective of this research was to establish the iron species generated during EC with iron anodes. Experimental results demonstrated that Fe2+, not Fe3+, was produced at the iron anode. Theoretical current efficiency was attained based on Fe2+ production with a clean iron rod, regardless of current, dissolved-oxygen (DO) level, or pH (6.5-8.5). The Fe2+ remaining after generation and mixing decreased with increasing pH and DO concentration due to rapid oxidation to Fe3+. At pH 8.5, Fe2+ was completely oxidized, which resulted in the desired Fe(OH)3(s)/ FeOOH(s), whereas, at pH 6.5 and 7.5, incomplete oxidation was observed, resulting in a mixture of soluble Fe2+ and insoluble Fe(OH)3(s)/FeOOH(s). When compared with Fe2+ chemical coagulation, a transient pH increase during EC led to faster Fe2+ oxidation. In summary, for EC in the pH 6.5-7.5 range and at low DO conditions, there is a likelihood of soluble Fe2+ species passing through a subsequentfiltration process resulting in secondary contamination and inefficient contaminant removals.

Arsenic in the breast milk of lactating women in arsenic-affected areas of West Bengal, India and its effect on infants.

Two hundred and twenty-six breast milk samples were collected from lactating women from 3 blocks of North-24 Paragans, one of the arsenic-affected districts of West Bengal, India. Out of 226 samples, only in 39 samples arsenic was detected. Urine, hair, and nail samples were also analyzed to know the arsenic body burden of the lactating women. Arsenic in drinking water was also analyzed. Principle component analysis (PCA) revealed that hair and nail arsenic was highly correlated with water arsenic concentrations, whereas arsenic in urine and breast milk did not cluster with water arsenic. Our present study indicated that among the lactating women who had high arsenic body burden and arsenical skin lesions, they had elevated level of arsenic in their breast milk. Arsenic in hair, nails, and urine samples of infants were analyzed, and the results showed significantly high-body burden of infants in those areas. PCA showed the age-dependent relationship between the hair and nail arsenic concentrations of the mothers and their babies.

Influence of sulfide (S2-) on preservation and speciation of inorganic arsenic in drinking water.

Generally, H2SO4, HNO3, HCl or the combination of ethylenediaminetetraacetate with acetic acid (EDTA-HAc) have been used to preserve arsenite and arsenate species prior to analysis. When these acidic preservatives are added in sulfidic water, instantaneous precipitation of poorly crystalline orpiment, As2S3(am), occurs, thereby lowering the total arsenic, As(Tot), analysis. A new method for the determination of As(Tot) was developed in which acid-preserved sulfidic water samples were oxidized with NaOCl, converting As2S3(am) and thioarsenic species to arsenate. A new method was also developed for the separation of uncharged arsenite and charged thioarsenic species in fresh, unpreserved sulfidic water by adsorbing the charged thioarsenic species while allowing uncharged arsenite to pass through a strong-base resin unhindered. The adsorbed thioarsenic species could be eluted efficiently with 0.16 M NaOCl solution.

Preservation of inorganic arsenic species in groundwater.

The objective of this research was to develop a robust preservation method for stabilizing inorganic As(IlI/V) species in synthetic and actual groundwaters. Ethylenediaminetetraacetic acid (EDTA), H2SO4, H3PO4, and EDTA-acetic acid (HAc) were evaluated in synthetic groundwater containing 3 mg/L Fe(ll) in the pH range 6.5-8.4 and Eh range -100 to +200 mV at room temperature. In the absence of strong UV light, only EDTA-HAc was found to be an effective preservative under all the experimental conditions tested. A total of 89 samples (including 16 samples in triplicate) from 55 drinking waterwells in Minnesota, California, and North Dakota were preserved with a combination of EDTA-HAc and speciated to evaluate its effectiveness for preserving inorganic arsenic species in actual groundwater samples. The preserved and field-speciated samples were repeatedly speciated and analyzed in the laboratory for up to 85 days after collection. Field-speciated As(lll) concentrations were compared with the As(lll) concentrations in the preserved samples. The results were highly correlated (slope 0.9773, R2 = 0.9986), which indicates that during sample transportation and storage the distribution of arsenic species did not change for samples preserved with EDTA-HAc.

Arsenic and other elements in hair, nails, and skin-scales of arsenic victims in West Bengal, India.

For the first time, biological tissues (hair, nails, and skin-scales) of arsenic victims from an arsenic affected area of West Bengal (WB), India were analyzed for trace elements. Analysis was carried out by inductively coupled plasma-mass spectrometry (ICP-MS) for 10 elements (As, Se, Hg, Zn, Pb, Ni, Cd, Mn, Cu, and Fe). A microwave digester was used for digestion of the tissue samples. To validate the method, certified reference materials--human hair (GBW 07601) and bovine muscle (CRM 8414)--were analyzed for all elements. The W test was used to study the normal/log normal distribution for each element in the tissue samples. For hair (n=44) and nails (n=33), all elements show log-normal distribution. For skin-scale samples (n=11), data are not sufficient to provide the information about the trend. Geometric mean, standard error, and range for each element were presented and compared with literature values for other populations. This study reveals the higher levels of toxic elements As, Mn, Pb, and Ni in the tissue samples compared with available values in the literature. The elevated levels of these toxic metals in the tissues may be due to exposure of these elements through drinking water and food. The correlations of Mn and Ni with other essential elements, e.g. Fe, Cu, Zn, suggest that Mn and Ni may substitute for those elements in hair, nails, and skin-scales. However, correlation represents the relation between two elements only and does not take into consideration of the presence of other elements. Principle component analysis was applied to explain the behavior among the elements present in hair and nails. This study reveals that in the arsenic-affected areas of WB, the concentrations of other toxic elements in drinking water and foodstuff should be monitored to evaluate the arsenic poisoning.

An automated sequential injection analysis system for the determination of trace endotoxin levels in water.

A new automated instrument based on the Limulus amebocyte lysate (LAL)-chromogenic substrate kinetic assay for the determination of bacterial endotoxins is reported. A computer controlled syringe pump-multiport valve combination was used to aspirate the sample and other reagents into a holding coil. The syringe was always filled with air; liquid did not enter the syringe. The valve could address up to four individual radial paths, fully referenced optical fiber LED-based absorbance detectors that were housed collectively in a single, small (20 x 20 x 30 mm) metal block with a heater and temperature sensor, and maintained at 37 degrees C. Assay mixtures containing sample or standards were delivered to the individual detector cells for the simultaneous collection of the time based absorbance data. The automated system determined endotoxins with good accuracy and reproducibility in the range of 0.005-0.5 endotoxin units (EU)/mL (r2 > or = 0.99). Based on three times the standard deviation of the blank and the slope of the calibration curve, the lower limit of detection was < or = 0.003 EU/mL. The variability of the assay method is less than 5% (n = 10). Analysis time required for a 0.005 EU/mL standard was <100 min. Appropriately diluted tap water samples were simultaneously analyzed by the present system and a manually loaded commercial microplate reader based instrument; the data were statistically indistinguishable at the 95% confidence level.

A continuous analyzer for soluble anionic constituents and ammonium in atmospheric particulate matter.

A new continuous soluble particle collector (PC) that does not use steam is described. Preceded by a denuder and interfaced with an ion chromatograph, this compact collector (3 in. o.d., approximately 5 in. total height) permits collection and continuous extraction of soluble components in atmospheric particulate matter. The PC is mounted atop a parallel plate wetted denuder for removal of soluble gases. The soluble gas denuded air enters the PC through an inlet. One version of the PC contained an integral cyclone-like inlet. For this device, penetration of particles as a function of size was characterized. In the simpler design, the sampled air enters the PC through a nozzle, and deionized water flows through a capillary tube placed close to the exit side of the nozzle by Venturi action or is forcibly pumped. Some growth of the aerosol occurs in the highly humid mist-chamber environment, but the dominant aerosol capture mechanism involves capture by the water film that forms on the hydrophobic PTFE membrane filter that constitutes the top of the PC and the airflow exit. Water drops coalesce on the filter and fall below into a purpose-machined cavity equipped with a liquid sensor. The water and the dissolved constituents are aspirated by a pump onto serial cation and anion preconcentrator columns. NH4+ captured by the cation preconcentrator is eluted with NaOH and is passed across an asymmetric membrane device. NH3 diffuses from the alkaline donor stream into a deionized water flowing countercurrent; the conductivity of the latter provides a measure of ammonium. The anions on the anion preconcentrator column are eluted and measured by a fully automated ion chromatography system. The total system thus provides automated semicontinuous measurement of soluble anions and ammonium. With a 15 min analytical cycle and a sampling rate of 5 L/min, the limit of detection (LOD) for ammonium is 8 ng/m3 and those for sulfate, nitrate, and oxalate are < or = 0.1 ng/m3. The system has been extensively field tested.