Application of Lean Six Sigma Methodologies and In-Vitro Dissolution Studies for Simultaneous Determination of Cefdinir and Sodium Benzoate by RP-HPLC and UPLC Methods in their Dosage Forms

Application of Lean Six Sigma and Quality tools plays an important role in interpretation of the data of quality attributes, decreasing waste time, remission manpower and reducing variation which will give intensity, confidence, and thoroughness. Thus, one can follow up and assure that the Process Capability Index (Cpk) is >1.33 and still capable during the all stages of the product. The purposes of this work are: to develop and validate a simple, accurate, precise and new RP-HPLC and UPLC methods, after reconstitution, accelerated stability and comparative in vitro dissolution studies. These were proceeded for the simultaneous determination of cefdinir (CFR) and sodium benzoate (SDM), respectively in the generic product; Dinar 125 mg / 5 mL powder for oral suspension (POS) and Dinar 300 mg capsule (CAP) and consequently it had been considered equivalent to the innovator product; Omnicef 125 mg / 5 mL POS and Omnicef 300 mg CAP using FDA media of 0.05 M phosphate buffer having pH 6.8. The chromatographic separation and resolution were performed at ambient temperature using:


Introduction
Recently, the use of application of quality tools and lean six sigma methodologies are widespread in most of the industries, especially pharmaceutical industries that are intended to reduce waste time, eliminate defects in the process and increase the capability of the process [1]. Simultaneous determination of binary mixture analytes in one reaction system using the same reagent and columns has become fascinating feature and very useful in modern analytical chemistry. This leads to saving money and effort of analysts as well as it is beneficial for quality control laboratory to analysis and Cefdinir is a white to slightly brownish-yellow solid. It is slightly soluble in dilute hydrochloric acid and sparingly soluble in 0.1 M pH 7.0 phosphate buffer. The empirical formula is C 14 H 13 N 5 O 5 S 2 and the molecular weight is 395.42 [2]. CFR belongs to a class of antibiotics called cephalosporins, which work for killing bacteria or halting their growth. CFR is prescribed to treat a wide range of bacterial infections, including certain types of pneumonia, sinusitis, bronchitis, strep throat, sore throat, middle-ear infections, and certain skin infections [2]. SDM is the chemical benzoate of soda (C 7 H 5 NaO 2 ) (Figure 1b), produced by the neutralization of benzoic acid with sodium bicarbonate, sodium carbonate, or sodium hydroxide. The salt doesn't occur naturally. The ingredient is used as an antimicrobial and as a flavoring agent used in food at levels not to exceed good manufacturing practice. Current usage results in a maximum level of 0.1 percent in food [3]. An HPLC method for determination of CFR is officially reported in the United States Pharmacopeia (USP) [4]. It is still a limited number of analytical methods that are reported for the determination of CFR individually or in combination with other drugs including High performance liquid chromatography (HPLC) [5][6][7][8][9][10][11][12], Ultra performance liquid chromatography (UPLC) [13], capillary electrophoresis and electrochemical methods [14,15], thin layer chromatography (TLC) [16], colorimetric and spectrophotometric [17][18][19][20][21], and comparative in vitro dissolution studies for cephalosporins [22,23]. Like CFR, SDM is officially reported in British Pharmacopeia (BP) [24] and United States Pharmacopeia (USP) including titration and HPLC. The literature review revealed that a variety of analytical methods have been reported for its determination in its pure drug and dosage forms using HPLC [25][26][27], Ultra performance liquid chromatography (UPLC) [28,29], HPTLC [30], Isoabsorption [31], and spectrophotometry [32,33]. For the best of our knowledge, there is no RP-HPLC and UPLC methods for the simultaneous determination of cefdinir and sodium benzoate in their pure, powder for oral suspension and capsule dosage forms without previous separation. Therefore, the main aim of this work is to establish this needed novel RP-HPLC and UPLC methods for the determination of binary mixture of CFR and SDM and application of lean six sigma methodologies, quality tools and comparative in vitro dissolution studies.

Apparatus
Waters ACQUITY ® Arc™ UPLC System, a quaternary liquid chromatography provides plug-and-play method compatibility

Solvent
Dissolve about 3.4gm potassium dihydrogen phosphate and 10.65gm disodium hydrogen phosphate in about 750 mL water.
Adjust the pH to 7.0± 0.05 using phosphoric acid or 1N NaOH solution then add sufficient water to make 1000 mL.

Standard Solutions
Stock Standard Solution (1500 μg/mL of CFR and 1000 μg/mL

SDM)
Accurately transfer 150 mg of CFR and 100 mg of CFR into 100   Waters CORTECS ® C18 column (50 mm × 4.6 mm, 2.7 μm particle size) at flow rate 0.3 mL/min, injection volume 0.3 μL was used for RP-UPLC where the total run time was 6 min.

Comparative in Vitro Dissolution Study
The main reason for making comparative in vitro dissolution study is to disclose any variation occurring in the final product market as change in inactive ingredients, change in raw material suppliers (as adding new supplier) or submission re-registration stability file product for ministry of health (MOH), so in the previous cases comparative in vitro dissolution study must be performed to release the drug products.

Test Preparation
Place one unit in each vessel from the generic product and the innovator one, using sinker in case of capsule and immediately operate the apparatus at the rate specified. The automated system will withdraw the samples at time intervals, then drug release was measured using the chromatographic system.

After Reconstitution Studies and Accelerated Stability
Re-constitution stability study was conducted on the first new

Process Capability Six-pack Quality Tools for Normally Distributed Assay
Lean six sigma and quality tools play an important role regarding assessing and estimating change during the process, and thus can be useful, also to choose or modify the process in some of the stages of the product improvement, where current work is used to analyse the ability of comparison among various suppliers and follow-up the process during the past two years and assure that the Process Capability Index (Cpk) is > 1.33.

Results and Discussion
The present work discusses comparative in vitro dissolution studies for simultaneous determination of the co-formulated binary mixtures of CFR and SDM in their suspension and capsule dosage forms which will lead to a paradigm shift towards pharmaceutical analysis where it reduces wasted time, effort, reagents, and columns. Also, application of lean six sigma and quality tools will help the quality team to detect the defect and variation during the process, so that they can be eliminated easily before the end of the process. Several trials were developed to optimize affecting parameters and to obtain a good separation of the binary mixture of CFR and SDM. So different compositions of mobile phases with different ratios were tried such as methanol (100%); methanol:  [35]. The acceptance criteria of the dissolution for product release were estimated according to international guidelines and USP monograph comparison. In vitro dissolution profiles of drugs were recommended on three different dissolution mediums (pH 1-7.5) In case of the monograph is not available [36]. The best developing system was 900 mL 0.05 M phosphate buffer, pH 6.8 Type II (paddle) at speed rate of 50 rpm with time intervals; 10, 20, 30 and 45 minutes. In the present study, the percentage of drug released for dissolution media were >80% in 10 min. According to the FDA Guidance for Industry and EMA guidelines, for dissolution profiles to be considered similar as shown in Figure 4, where more than 85% of the drug is dissolved within 15 minutes, dissolution profiles may be accepted as similar without further mathematical evaluation of the similarity factor f2 [37].

Method Validation
The proposed method was validated, in accordance with ICH guidelines [38]. Regarding Linearity and range, LOD and LOQ, Accuracy and recovery, Precision (repeatability and intermediate precision), Robustness, Stability of the analytical solution, Formulation assay, system suitability, specificity.

Linearity and Range
In order to evaluate the linearity of an assay procedure, a series of standards at different concentrations of the target concentration were prepared. After running each preparation in triplicate, a linear regression analysis was performed on the average peak areas versus the concentrations of the levels studied. The linearity of the proposed methods was obtained over concentration range (50 -1000 and 5-50 µg/mL) with coefficient of regression >0.999 for CFR and SDM, respectively. The linearity results are shown in Table 1.

Limit of Detection and Quantitation
The limit of detection (LOD) and limit of quantitation were based on the standard deviation of the response and the slope and can be calculated as LOD=3.3×σ /slope and LOQ=10×σ /slope, where σ = the standard deviation of the response as listed in Table   1.

Accuracy and Recovery
The recovery was calculated in triplicates of three Results are shown in (Table 2). Moreover, accuracy was evaluated by applying the standard addition technique to the samples with good recovery suggesting that there is no involvement from excipients as shown in Table 3.  were obtained as presented in Table 4.

Robustness
For measuring the capability of the method to remain unaffected by small, but deliberate, variations in method's parameters such as: influence of variations of pH of the mobile phase (±0.2), Flow rate change (±0.1 mL/min), wave length change (254±2.0nm) and column temperature change (30,25˚C). Good results were gained as presented in Table 4.

Stability of the Analytical Solution
The method was proceeded to evaluate the stability assay of the standard solution when it was stored in a fridge and another part was stored at room temperature for 24 hours, then the solutions were tested against a freshly prepared standard; relative standard deviation should be less than 2%. Results are displayed in Table 4.

Formulation Assay
The proposed method was performed by assaying six samples from the commercially dosage form (OMNICEF ® , DINAR ® 125 mg /5 mL POS and OMNICEF ® , DINAR ® 300 mg CAP) to determine the assay of CFR and SDM. The obtained results are displayed in Table   5. indicating that the method is selective for the analytes without interference from the excipients used to formulate.

System Suitability
Tests were based on the concept that chromatographic system, analytical operations and samples to be analyzed constitute an integral system that should be checked by calculating various parameters such as the number of theoretical plates (N), tailing factor (T), resolution (Rs), precision and selectivity (k') to ensure the performance of the system. All calculated parameters were found within the acceptable limits indicating good selectivity of the method as listed in Table 6.  Table 3 indicate no matrix interference.

Application of Comparative in vitro Dissolution Study for the Proposed Method
In vitro dissolution was carried out on two generic products viz., DINAR 125mg/5mL POS and DINAR 300 mg CAP dosage forms selecting dissolution parameters as described by FDA dissolution methods [39]. For comparative studies, the in vitro dissolution of innovator brand e.g., OMNICEF 125mg/5mL POS and OMNICEF 300 mg CAP dosage forms were performed at the same conditions and results were compared with those of generic brands. The quantitative release of drug at specified intervals from the product was determined using the validated HPLC and UPLC methods. The dissolution results which are summarized in Table 7 showed that 9/13 the value of RSD % is less than 10 % at the initial point and less than 5 % for other intervals. Also, the in vitro dissolution profile and the cumulative percentage of generic and innovator products released were plotted against time (Figure 4a & 4b), The dissolution profile of generic brand showed similar behavior to the innovator one.
There was an agreement that the f2 test is not necessary when the two products each provide at least 85% dissolution in 15 min. The percentage dissolved for all products was above 85% of the labeled claimed content from the very first sampling time.

Application of Quality Control and Statistical Tool for Normally Distributed Assay
Data was collected for the last two years of 100 batches of assay for CFR as mentioned in Table 9 by our quality team for monitoring the manufacturing operation for different suppliers. Then, were measured using the Minitab program. Process capability six-pack analysis indicated that the data for the first supplier API corporation (Japan) is statistically capable and more accurate than the second supplier as Cpk is 1.83 (Figure 6a & 6b), means that it meets 6 sigma levels. Also, in the second supplier covalent lab (India) the process is normally distributed, within the control specification and statistical control as the Cpk is 1.47 i.e., it meets 4 sigma levels.