Simultaneous Determination of Piroxicam and Codeine Phosphate Hemihydrate in a Pharmaceutical Dosage Form Using Validated HPLC Method

An easy, eclectic, precise high-Performance Liquid Chromatographic (HPLC) procedure was evolved and validated to estimate of Piroxicam and Codeine phosphate. Chromatographic demarcation was accomplished on a C 18 column [Use BDS Hypersil C 18 , 5μ, 150 x 4.6 mm] using a mobile phase of methanol: phosphate buffer (60:40, v/v, pH=2.3), the flow rate was 1.1 mL/min, UV detection was at 214 nm. System Suitability tests (SSTs) are typically performed to assess the suitability and effectiveness of the entire chromatography system. The retention time for Piroxicam was found to be 3.95 minutes and 1.46 minutes for Codeine phosphate. The evolved method has been validated through precision, limit of quantitation, specificity, limit of detection linearity and accuracy. (LOD) was 1.92  g/mL and (LOQ) was 6.336  g/mL for Piroxicam, whereas (LOD) for Codeine phosphate was 0.29  g/mL and (LOQ) was 0.95  g/mL. Piroxicam and Codeine phosphate showed a linear signal in the domain of 5-50 µg/mL for each compound. This research presided to evolve and validate an HPLC method, and the proposed procedure can be used to estimate these drugs in their combined dosage forms.


Introduction:
Piroxicam (Fig. 1) is a drug classified as a non-steroidal anti-inflammatory drug (NSAID) having an antipyretic and analgesic characteristic. It is used to treat rheumatic diseases such as inflammation, pain from injury, menstrual cramps, arthritis, musculoskeletal disorders, and nonrheumatic diseases such as biliary and ureteral colic, dysmenorrhea inflammation and fever 1 . Piroxicam has an oxime group and N-heterocyclic carboxamide. Piroxicam has an IUPAC name (4-Hydroxy-2-methyl-N-(2-pyridinyl)-2H-1,2benzothiazine-3-carboxamide 1,1-dioxide), it is white to light yellow, crystalline powder with a molecular weight of 331.35 g/mol and practically insoluble in water 2,3 , Codeine Phosphate (Fig. 2) is an opioid derived from the immature poppy seed plant (Papaver somniferum). It has a phenolic hydroxyl group with a methyl substitution and has a morphine-like structure. The chemical description of the phosphate salt form is 7,8-Didehydro-4,5 alpha-epoxy-3-methoxy-17-methylmorphinan-6 alpha-ol phosphate hemihydrate and the molecular form is C 18 H 24 NO 7 P·½H 2 O. Codeine has one chiral center. Codeine has a molecular weight of 406.4 g/mol 4 . Literature reviews do not reveal any analytical techniques for the determination of Piroxicam and Codeine Phosphate as a combination in Pharmaceutical Dosage Forms, but they reported some analytical methods to estimate Piroxicam that include: spectrophotometric method 5-8 , highperformance liquid chromatographic method [9][10][11][12] and capillary electrophoresis 13

Instrumentation and Chemicals
A Shimadzu Prominence SPD-20A HPLC PDA, Japan and an auto sampler (Shimadzu, Japan, model SIL-10AD) and a model SPD-20AV UV-VIS detector were used for chromatographic measurements. Working standards Piroxicam (99.5% w/w) and Codeine Phosphate (99.3% w/w) were kindly gifted by UNIPHARMA and ULTAMEDICA respectively (a Syrian pharmaceutical companies in the Damascus countryside, Syria). Fixed-dose combination tablets that contain 10 mg of Piroxicam and 10 mg of Codeine Phosphate Hemihydrate were formulated in the Industrial Pharmacy Department, Faculty of Pharmacy, Damascus University, Syria. Disodium hydrogen phosphate, monosodium dihydrogen phosphate, orthophosphoric acid, potassium dihydrogen phosphate and triethylamine (analytical grades) were gifted by MEDICO labs (a Syrian pharmaceutical company in the Homs countryside, Syria). Methanol (HPLC grade) were purchased from J.T. Baker (USA).

Preparation of Standard Stock and Working Solutions
Preparation of methanolic hydrochloric acid 0.01 N: It is prepared by diluting 0.9 mL of hydrochloric acid with methanol to a volume of 1 L 15 .
Preparation of phosphate buffer solution pH=6.8: Dissolving 8.722 g of disodium hydrogen phosphate (Na2HPO4.2H2O) and 7.038 g of monosodium dihydrogen phosphate (NaH2PO4.H2O) in sufficient water to produce 1000 mL pH adjusted to 6.8 with ortho-phosphoric acid.
Preparation of potassium phosphate buffer solution pH=2.3: Dissolving 2.04 g of potassium dihydrogen phosphate (KH 2 PO 4 ) in sufficient water to produce 1000 mL, then add 2 mL of triethylamine (TEA). pH adjusted to 2.3 with orthophosphoric acid 16 .
Preparation of Standard Solutions: Piroxicam stock solution (0.1 mg/mL) and Codeine phosphate stock solution (0.1 mg/mL) were prepared in methanolic hydrochloric acid 0.01 N. Mixed standard stock solution containing 50 µg/mL of Piroxicam and 50 µg/mL of Codeine phosphate was prepared by diluting the standard stock solution in methanolic hydrochloric acid 0.01 N.

Analytical Conditions of HPLC Method
The HPLC system (Shimadzu Prominence HPLC, Japan) consisted of a Pump that was set to inject 20 l per injection at once. The detector (SPD-20A PDA) consisted of UV/ VIS in the one hundred ninety to seven-hundred nm range which can be used for all UV analyses. BDS Hypersil C 18 analytical column (150 × 4.6 mm, 5µ) (stationary phase) was utilized for LC separations. The mobile phase contained methanol: phosphate buffer pH=2.3 (6:4, v/v), and ortho-phosphoric acid was used to adjust pH to 2.3. Degassing then filtering procedures were performed on the mobile phase before using it. The pump was set at a flowing rate of 1.1mL/min. All measurements were accomplished at ambient temperature with a detection wavelength of 214 nm.

Validation of the HPLC Method
System Suitability test should be performed before starting validation of the HPLC method where the standard solutions of the two drugs are injected sundry times and the relative standard deviation is calculated, which should be less than %2 at all.

System Suitability Test (SST)
SST is particularly fulfilled to decide the column efficiency, resolution, and repeatability of a specific chromatographic system to affirm its ability for a described evaluation. SST is an important characteristic of all HPLC analytical systems. When the system suitability for the evaluation method uses the high-performance liquid chromatography, the subsequent parameters are had to be in the appropriate outlines 17 : Retention Time (Rt): It is the interval time between the injection of the sample into the tool and the advent of the maximal peak at the detector. 18 Capacity Factor: It expresses the overlap of the injected sample material with the column filling and the mobile phase and has to be more than 2 18,19 . Resolution: It demonstrates the dissociation power of the whole chromatographic system to the specific compounds of the commixture. It is expressed as the proportion of the range between peaks to the peak width value. If R is identical to or greater than one then components are absolutely separated. If R is much less than one, then components are overlapped 18 . Theoretical plates: The quantity of theoretical plates is a degree of the overall performance of the column and has to be more than 2000 17 . The HPLC procedure was established with appreciation to the subsequent Parameters according to the ICH guidelines 20 . Linearity: The capacity of the analytical method to offer measurement outcomes immediately proportional to the concentration of the analyzed material withinside the sample in the given range, either immediately or after performing precise mathematical transformations. The linearity of a standard series preparation withinside the range of 5-50 µg/mL has been determined. Linear standardization curves had been generated with the aid of using plotting the peak area in opposition to the concentration of the drug 21,22 . Accuracy: The HPLC procedure accuracy was carried out inside the procedure range, as follows: three weights of every drug was prepared to give concentration (10, 25 and 45) µg/mL. The percentage for every sample and the relative standard (RSD) was Calculated. The relative standard value should not be greater than 2% 23

Validation of HPLC Method System Suitability Test (SST)
The System suitability test was conducted by injecting six injections of resolution solution.
The dissociation was carried out by setting the pump at a flowing rate of 1.1 mL/min. The retention times for Piroxicam and Codeine phosphate were 3.95 and 1.460 minutes, respectively (Fig.3). Acceptable retention time (Rt), theoretical plates, tailing factor and good resolution for Piroxicam and Codeine phosphate were obtained as shown in Table  1.   (Fig. 4). A prime correlation existed between the peak areas and concentrations of Piroxicam and Codeine phosphate was presented in Table 2.

Accuracy:
The accuracy test affirmed appropriate retrievals % with small (RSD%) against concentrations. The outcomes signify that the procedure is highly precise for simultaneous estimation of the mentioned drugs, as shown from the data in Table  3.  Specificity: The specificity was realized by completing the demarcation of Piroxicam and Codeine phosphate peaks in the existence of the tablet excipients (Fig. 6). No overlap was noticed due to any indeterminate excipients of the formulated tablet at the retention times of Piroxicam and Codeine phosphate. (The used excipients were sodium starch glycolate, Crosspovidone-XL and crosscarmellose sodium), (Fig. 5).  Table 2.

Analysis of the Drugs in the Tablets (Assay):
Utilizing the proposed analytical procedure, assays of Piroxicam and Codeine phosphate in their tablets were accomplished. Satisfying outcomes were found for the two drugs in prime adjustment with the mentioned amounts proposing the appropriation of the procedure (Fig.  3). The retrieval % ± RSD of five duplicate outcomes were 99.41 ± 0.53 for Piroxicam, 100.3 ± 0.62 for Codeine phosphate. Table 6.

Conclusion
The proposed HPLC method demonstrates an easy, precise and reproductive method of quantitative analysis for concurrent estimation of Piroxicam and Codeine phosphate in bulk and tablet dosage forms. The HPLC method was validated according to ICH guidelines, and it was distinctive and there is no overlap from any of the sample compounds. It was deduced that the evolved procedure provided numerous profits such as speedy, slight-effective, easy mobile phase and making steps, innovated sensitivity and comparative low run time made it specific, dependable and effortlessly reproductive in any quality control setup imparting all of the parameters are accompanied appropriately for its meant use.