It is important to distinguish a drug with a characteristic that poses a high occupational risk from its characteristic therapeutic effect & benefit, in short potency.
 
For example, a drug that increases the acceptance of a transplated organ is given to patients in high doses. However, occupational exposure of that same drug may cause birth defects in the off-spring of workers who may come into contact with it. Therefore OEL (Occupation Exposure Levels) will play a major role rather than plain potency of the api.
 
If one is able to establish effectiveness of manufacturing environment containment systems, waste stream management and industrial hygiene (IH) practises performed with respect to OEL, further complemented by cleaning validation data, should by far indicate compliance.

Definitions and scope of quality systems in pharmaceuticals has evolved over a period of time. The “Thalidomide babies tragedy” prompted the concept of continuous or cGMP. With cGMP came into existence the concept of Quality Assurance or “Zero defect”. QA advocated that quality cannot be created at the end of processing, but has to be in-built into a product at every step of manufacturing process.

Further improvements in quality systems throughout the 1990’s and beyond brought about concepts of internal audits, documentation and validations. Y2K improved quality systems further...introduced 21CFR part 11 compliance measures. Year 2010 and beyond promises further refinement in quality systems-  Quality-by-Design (QbD).

What is quality by design?

Quality by design means designing and developing a product and associated manufacturing processes that will be used during product development to ensure that the product consistently attains a predefined quality at the end of the manufacturing process.

Where to implement quality by design?

Quality by design implementation targets the following departments within a pharmaceutical company-Management, Procurement, R&D, Manufacturing, Testing, Quality control, Quality assurance, Regulatory, Logistics, Sales, Warehouse/ Supply chain including vendors facilities, CRO and CMO.

Principles of quality by design?

QbD scope assume that problems can be anticipated and their occurrence prevented by reviewing data and analyzing risks associated with operational and quality system processes and by keeping abreast of changes in scientific developments and regulatory requirements. The central goal of a quality system is the consistent production of safe and effective products and ensuring that these activities are sustainable. A robust quality system will promote process consistency by integrating effective knowledge-building mechanisms into daily
operational decisions. When fully developed and effectively managed, a QbD system will lead to consistent, predictable processes that ensure that pharmaceuticals are safe, effective, and available for the consumer.

Framework of quality by design?

Quality by design integrates quality systems and risk management approaches into its existing programs with the goal of providing the necessary framework for implementing quality by design (building in quality from the development phase and throughout a product’s life cycle), continual improvement and risk management in the drug manufacturing process and also for post development changes and optimization.

Quality risk management (governed by CAPA- corrective actions preventive actions) is a valuable component of an effective quality systems framework. Quality risk management can, for example, help guide the setting of
specifications and process parameters for drug manufacturing, assess and mitigate the risk of changing a process or specification, and determine the extent of discrepancy investigations and corrective actions.

CAPA focuses on investigating, understanding, and correcting discrepancies while attempting to prevent their recurrence. QbD system models discuss CAPA as three separate concepts, all of which are used in this guidance: 

1. Remedial correction of an identified problem.
2. Root cause analysis with corrective actions to help understand the cause of the deviation and potentially prevent recurrence of a similar problem.
3. Preventive action to avert recurrence of a similar problem.

 Review outcomes typically include:

• Improvements to the quality system and related quality processes.
• Improvements to manufacturing processes and products.
• Realignment of resources.

The results of a management review would typically be recorded. Planned actions should be implemented using effective CAPA and change control procedures.


To be continued...

Isolation and identification of bacteria-

The isolation was done by transferring the colonies from nutrient agar plated to sterile enrichment broth media. These included MacConkeys broth (for detection of Staph.aureus) and Cetrimide broth (detection of P.aeruoginosa). The specimen tubes were incubated for 48 hours at 37 + 1 oC in inverted position for
24 hours and 507 days.

Allocation of mannitol salt agar B30-

The colonies grown on Vogel –Johnson’s medium was streak plated on sterile mannitol –salt agar medium for confirmation regarding presence/ absence of Staph. aureus . Pure staphylococcus culture ATCC 12600 was simultaneously streaked on separate plates to serve as positive control.

Further study of culture on selective media-

The colonies grown on Vogel- Johnson’s medium and MacConkeys medium were tested for Gram reactions and motility characteristics.

Biochemical tests-

In order to further ensure that the organisms cultured on Vogel- Johnson’s and Mac Conkeys agar were probably E.coli, Staph. aureus or Salmonella species, the inoculums of the colonies from respective agar medium were employed in biochemical tests comprising of hydrolysis of starch, nitrate reduction, oxidase and catalase activity, liquefaction of gelatin, IMVic, Oxferm reaction etc. 
 
The contents of this article belong to extensive laboratory work done by me to assess microbial contamination on few brands of dye and lake colors available in the market.

EXPERIMENTAL-

Materials-

Marketed samples of indigo carmine, sunset yellow, erythrosine and ponceau 4R dye and their aluminum lakes. Materials used in the preparation of the lakes including aluminum hydroxide powder (batch nos. 103,374,AD3 and paste P61).  Nutrient agar, Vogel-Johnson’s medium, Mac Conkeys medium, Cetrimide medium and
agar media of these and mannitol salt agar were from M/s Hi media, India. Other stains and chemicals employed were of highest purity.

Methods-

Sampling, sample handling and sample condition-
 
All the samples obtained were powders, excepting aluminum hydroxide, which was supplied also as a paste. The samples were received in tightly closed glass bottles.

Sample treatment-

The surface of the sample container was disinfected with an aqueous mixture of 80 % alcohol v/v and 1 % v/v. The surface was dried with sterile gauze before opening and removing the contents in laminar air flow cabinet.

Microbial evaluation-

(I) Preparation of stock inoculums -

One-gram test sample was aseptically inoculated in 25 ml of sterile nutrient broth. The inoculated medium was kept undisturbed for about 15 minutes, to ensure complete transfer of microbes from sample to the medium. The clear supernatant liquid was then used a sample for performing the tests.

(ii) Determination of MPN by serial dilution method-

One ml of stock inoculums prepared as mentioned under (i) was diluted with 9.0 ml nutrient broth to give a dilution of 1:10. this was further serially diluted with sterile nutrient broth to give 1:100, 1:1000 and 1: 10000
dilutions.

The labeled tubes were inoculated at 37 + 1 deg.C for 24- 48 hours and 5-7 days after which they were observed for the presence or absence of growth. The tube in which the growth was not clear from appearance was sub-cultured into fresh sterile nutrient broth tubes (0.1 ml was inoculated into 10 ml sterile nutrient broth). Negative and positive (E.coli ATCC 11775) control was maintained to test the effectiveness of sterilization procedures and growth promoting property of the nutrient media respectively.

(iii) Determination of bacterial count by plate count technique-

The stock inoculums prepared under (i) was serially diluted with sterile physiological saline to give 1:10,1:100,1:1000,1:10000 ,1:1,00,000 ,1: 10,00,000 dilutions. Depending on the MPN count values determined above in (1) 1.0 ml of the last two dilutions of the sample showing growth were pour plated using 15.0 ml of sterile nutrient agar. The plates were then incubated at 37 + 1 oC for 24 hours and 5-7 days and the MPN values were determined.

(iv) Detection of fungi-

One ml of the stock inoculums mentioned under (i) was added to 90 ml of Saborauds broth. Positive control (Candida albicans ATCC) and negative controls were maintained side by side. The tubes were incubated at room temperature (RT) for a period of 7-15 days. 
 
In the next chapter, we shall discuss further on the experimental and isolation procedures of the microbes.

With the advent of out-sourcing of pharmaceutical products especially by Americas and the western world, pharma products have to meet international standards. This is not possible if quality is built into the product all the way through its trajectory from birth i.e. production development till marketing and administration to patients.

Therefore, all materials that get into a pharmaceutical product need to be closely scrutinized for its quality aspects. A commonly used excipients in formulation is color. Although, the percentages of color used in pharma products is quite low, the effect of color on formulation stability or ability of coloring ingredient for destabilizing products is well known. 

Presence of microorganisms in large numbers in drugs and cosmetics preparations is undesirable since it may lead to spoilage of products. The product can change in color, consistency or manifest visible growth.
Furthermore, presence of microbial contaminants would constitute a potential hazard to public health although reports on contaminated cosmetics are rare.

A critical component involved in color stability is the microbial load. Some preliminary work on microbial contamination in cosmetics has been reported absent twelve years back from this laboratory. The earlier work in this field has been reported by Doren , Woodward , Riger , Wederburn, Kalings and Bruck . 

Various sources of contamination by microorganisms have been enumerated . Raw materials are likely sources of microbial contamination and should be examined microbiologically on a routine basis. Even IP, BP and USP have recognized this fact and have set standards for total number of aerobic bacteria in a variety of products for e.g. gelatin, aluminum hydroxide gel etc. A published report by FIP working party indicated presence of high levels of microorganism not only in substances of animal and vegetable origin but also of synthetic or purified
substances including talc, kaolin, vitamins, lactose etc.

Dye colors and their lakes used for improving the elegance of the medicines and cosmetic products could become a potential source of microbial contamination in these formulations. Although the colorants are evaluated for their toxicity, their safety as regards the microbial contamination has attracted very little
attention of the regulatory authorities. The methods for evaluation of medicinal preparations are given in official compendia while those for cosmetic preparations have been adequately described by Lucas (Ref 9-12).

With this background and the fact that, the regulatory authorities might include the limits of microbes in colorants work on lakes manufactured by one company has been undertaken. The contents of the case study disclosed here is authentic. This article is published here in to give an update of our research findings to the scientific community at large with out compromising in any way the confidentiality of the matter with all associated with this work.

Results and Discussion:

Identification test-

Mass, IR spectral data and DSC thermo gram indicated that the pseudoephedrine test sample had high purity and compared well with the reference standard.

Method validation-

Under the chromatographic conditions described, the migration distance obtained in the case of both the reference as well as the test samples of pseudoephedrine hydrochloride was 34.0 mm (Rt – 0.41). the reference standard sample showed a single band. The test for selectivity employing mixture of pseudoephedrine HCl and phenylephrine showed sharp separation of HPTLC plate in the solvent system containing formic acid as mentioned earlier and compared well with the respective reference standard. The migration distance of
pseudoephedrine HCl and phenylephrine were 34.0 mm (Rt – 0.41). and 51.0 mm (Rt – 0.62). respectively.

Observation on the limits of detection and quantification, selectivity, linear range and ruggedness of the proposed method were as follows-

Limit of detection (μg) 5
Limit of quantification (μg) 10
Linear range (μg), regression coefficient, r= 0.999 35-140
Sensitivity (units) 11.2
Ruggedness (%CV) between analysts 1.224
Ruggedness (%CV) between laboratories 2.32

Comparing the accuracies (%CV calculated over the linear range) of the HPLC and HPTLC method, It was found that the accuracy of the former was 1.362 units, while that of the latter was 3.002 units.

Application to stability studies-

The results of the stability studies indicated that there was probably no interference of degradation products during the assay. The degradation products however, were not identified since the reference standards for the same were not available.


CONCLUSION-

A simple, selective and rugged HPTLC method has been developed and validated for determining pseudoephedrine HCl powder. The proposed method is comparable to the HPLC method (reported earlier in the literature). The HPTLC method can be applied for studying the stability of the pseudoephedrine HCl and sulphate samples stored under stress conditions of temperature and humidity. The method further needs to be tried for the analysis of pseudoephedrine in solid and liquid dosage forms.

Method validation: selectivity, limit of detection, limit of quantification, linearity range and sensitivity-

The selectivity of the method was checked by spotting 50μg x μl –1 of pseudoephedine HCl (a structurally
related drug) individually and as a mixture (in 1:1 proportion) on a plate. Rest of the procedure followed was a described above under, “preparation of calibration curve”. The limit of detection, limit of quantification, linearity
range of the calibration curve and sensitivity (which is the slope of the calibration curve) were also determined

Accuracy-

Accuracy of the assay was determined following standard procedure; the results obtained by spotting known amount of drug applied on the plate were compared with the experimental values. The accuracy was expressed in terms of % basis using the following equation-

% Bias = Observed value – Original concentration
-------------------------------------------------------------------------- x 100
 Original concentration

 
Precision-

The inter- day assay variability was calculated for all concentrations in the linearity range (35,70,105 and 140 μg). The intra- day variation was determined on six replicate samples of a low (35 μg) and a high (140 μg) of the concentration curve.

Ruggedness-

Ruggedness of the analytical method was determined by the analysis of the same sample of pseudoephedrine HCl in two separate laboratories having similar equipments by three different analysts on consecutive days for a week. The coefficient of variation (%CV) amongst the results obtained was calculated.

Comparison of HPTLC with HPLC-

The accuracy of the proposed HPTLC method was compared with a reported HPLC method of estimation of pseudoephedrine HCl (detected at 210 nm) using Partisil SCX (10μm) HPLC column (25cm x 4.6 mm). The mobile phase comprised of 24 mM phosphate buffer (pH=2.3) in 50 % aqueous acetonitrile solution (1.5 ml per min.).

Applicability to stability studies-

The proposed HPTLC technique was applied in evaluating the stability of pseudoephedrine HCl samples stored under exaggerated conditions of temperature (40,50 and 60 + 1 deg.C) with relative humidity of 58,75 and 90 %. The standard samples (70 μg /ul) during the analysis were spotted after every 10 tests samples during the
analysis. The method was also applied in the analysis of aged pseudoephedrine sulphate samples stored under
similar conditions; the details of the stability studies are being published elsewhere.

Tomorrow, we shall take up discussion on results and discussion of these experiments.

Instruments-

Camag HPTLC system comprising of Camag IV sample applicator Linomat, Hamilton syringe 100 micro liter, Camag twin trough chamber (20x10 cm2), Camag scanner Ii V3.14 with ACTS software version 3.12, HPLC unit (Jasco PV 980) equipped with intelligent sampler (Jasco 851), Intelligent UV-Vis detector (Jasco model 875)
and integrator (Jasco model 870 IT), Mass spectrometer (Schimadzu QP 1000) equipped with EI mode, FTIR spectrophotometer (Jasco model FT/IR- 45300), Differential scanning calorimeter (Schimadzu model DT040 thermal analyzer equipped with chromatopac CR 6A integrator).

Drugs, chemicals and other materials-

Silica gel 60 F 254 HPTLC plates (20x 10 cm, Merck), pseudoephedrine hydrochloride USP, pseudoephedrine sulphate USP, (from a reputed Indian company), pseudoephedrine hydrochloride reference standard (B.Knoll, Germany), phenyl ephedrine reference standard (B.Knoll, Germany), ethylene dichloride and benzene (HPLC grade, Spectra Chem., India), toluene, ethyl acetate, cyclohexane, acetone, methanol and chloroform (HPLC, E.Merck India grade), potassium hydroxide GR and ammonia (Qualigens, India) and formic acid 98/100 % (BDH grade, India ). All the chemicals were used without further treatment. All volumetric glassware used for the study was calibrated.

Stock solution of pseudoephedrine hydrochloride (reference standard) 7 μg x ul –1 (A) was prepared by dissolving 70 mg of the material in degasses distilled water in a 10 ml volumetric flask.

Identification and purity of pseudoephedrine hydrochloride test sample-

Mass spectrum, IR spectrum and DSC thermo gram of pseudoephedine (test sample) were recorded using the instruments mentioned earlier and the data obtained were compared with those of the reference sample. Due to some difficulties, the impurity profile of the pseudoephedrine HCl could not be determined.


HPTLC of pseudoephedrine hydrochloride- Preliminary experiments-

Pseudoephedine HCl was spotted on HPTLC plate using Linomat IV. The plate was developed in a twin trough chamber. A single band was obtained indicating high purity of the test sample. However, the bands either failed to migrate from the base line or they almost migrated to the solvent front. Amongst all the reported systems employed including ethylene chloride – methanol- benzene (70:25:5 v/v/v) showed good movement of the band except that it showed tailing. Further studied on this has been presented here.

Preparation of calibration curve, sample application, plate development and densitometric evaluation-

Stock solution A was prepared as mentioned above 1,2,5,10,15 and 20 μl of this solution corresponding to 7,14,35,70,105 and 140 μg respectively of pseudoephedrine HCl were spotted as individual bands using Camag Linomt IV sample applicator equipped with a 100 μl Hamilton syringe. The plate was developed to a distance of 80-85 mm (35-40 min) in a solvent system consisting of ethylene dichloride- methanol-benzene- formic acid (14 :4:2:1 v/v/v/v) in a Camag twin trough chamber pres-saturated with the solvent system for a period of
not less than 45 minutes.

The developed plates were scanned densitometrically observing the following conditions: [I] lamp deuterium. [ii] Scanning speed 4.0 mm/s, [iii] slit dimensions 4x3 mm2, [iv] detection wave length 254 nm, [v] sensitivity 230 units and [vi] span 40 units.

The migration distance (MD) of the band was taken as the response standard during the scanning and integration of the developed bands. Peak area (AUC) was then plotted against the concentration values to obtain a calibration curve.

In the next chapter, we shall take up discussion on method validation and its results.

INTRODUCTION-

Analysis is a critical and integral part of the pharma business. Its only up on clearance of products on analysis can the products be even released into the market. Hence, systems of analysis as well as the analytical tools assume prime importance. Several well known analytical tools viz. HPLC, GC, IR, UV-Vis, atomic absorption spectrophotometer etc. are available to a pharmaceutical analyst. However, to my mind, pharmaceutical analyst has still not utilized all analytical techniques science has offered to us. To name few, are Near Infra red- NIR, Raman spectroscopy and HPTLC.

With the process analytical technique- PAT, claiming importance in quality control departments of several established pharmaceutical companies especially in the US, one has to rethink on easy yet accurate ways of content measurement in pharmaceutical products. HPTLC is another such easy analytical technique that  is precise, speedy and easy to work with but has not become very popular in the analytical field

To elucidate the merits of HPTLC, without promoting any brand of the analytical equipments, I have mentioned herein few cases (published in serial order). This work was done during my postgraduate days wherein we attempted to study use of HPTLC in the analysis of assay of active pharmaceutical ingredients- API, formulations and from plasma samples of a bio-equivalence study.

This article attempts to depict merits of HPTLC in estimation of assay of API. Pseudoephedrine hydrochloride is a model drug for this study.

Pseudoephedrine hydrochloride, a stereoisomer of ephedrine belongs to the class of sympathetomimetic drugs 1. Its main clinical use being a nasal decongestant. It is employed alone or in combination with other agents.

Analytical methods reported for estimation of pseudoephedrine include thin layer chromatography [TLC], gas chromatography [GC] and high performance liquid chromatography . However, quantitative high performance thin layer chromatography [HPTLC] estimation for this drug has not been reported hitherto.

The GC and HPLC assay procedure although, possess good sensitivity are tedious. TLC on the other hand is easy but lacks the sensitivity and selectivity necessary in the analysis of pharmaceutics. HPTLC possess the advantages of being simple, rapid (since 10 –20 sample can be estimated on a single plate), and easy to operate giving rapid performance with high resolution. The method has widely been accepted for qualitative and quantitative evaluation of drugs belonging to various classes viz. vitamins, steroids, amino acidic , alkaloids ,
antibiotics etc.

The objective of this study was (i) to develop HPTLC method for determination of pseudoephedrine and (ii) to apply the method to the quantitative estimation of pseudepherdine hydrochloride samples stored under stress conditions.

ii) Film studies- 
 
Cooper et al (1972) reported that an interaction between gelatin and dyes in the gelatin films could be studied by examining the shifts in λ max in thevisible range and for changes in the amide I (1625 cm –1) and amide II (1520 cm –

1 ) bands of the protein in the IR spectrum. IR spectra of gelatin films containing RO (at all the concentrations
attempted) , copper sulphate and potassium iodide showed disappearance of both these peaks indicating gelatin – mineral interaction. The dissolution data of the above stored films also indicated protracted disintegration. The IR spectra of the films containing manganese sulphate, magnesium oxide, dicalcium phosphate and zinc sulphate did not indicate interaction. The dissolution data (except in case of manganese sulphate) also supports this observation. It was observed that the rate of interaction accelerated due to-
 (1) increased humidity and (ii) presence of RO/ FF (except in case of manganese sulphate where iron salts showed a protective influence)
2) Liquid phase interactions
3) Changes in pH and viscosity.

The soluble salts used indicated acidic salts (copper sulphate and manganese sulphate) and basic (potassium iodide) while the insoluble salts included alkaline earth metal salts (magnesium oxide and dicalcium phosphate) and iron salts (RO/ FF). In case of acidic and basic salts, any interaction between gelatin and the action would bring forth changes in the pH. The decrease in pH with stored gelatin solutions containing copper sulphate and an increase in pH with those containing potassium iodide support interaction. The interaction possibly even increased the viscosity of the samples as observed from the viscosity data.

Insignificant change in the pH indicated lower degree of interaction (as also observed from atomic absorption spectral data given under formulation studies). Also, the insignificant changes in viscosity value supports this
statement.

CONCLUSION-

The minerals employed in sot gelatin hematinic capsules show migration into the gelatin shell due to the higher water and humectants content of soft gelatin capsules shell than the hard gelatin shell which subsequently end up in interaction between the gelatin and the captions resulting in protracted disintegration or rupture. These interactions in the capsules could lead to noncompliance with the official disintegration tests standards. However, the effect of this interaction on the bioavailability of the content has not been reported so far.