INTRODUCTION:

Paracetamol:

N-(4-Hydroxyphenyl) acetamide or Paracetamol (Par) is a white or almost white crystalline powder. Sparingly soluble in water, freely soluble in alcohol, very slightly soluble in methylene chloride¹, fig. 1,a.

(Par) is a p-aminophenol derivative that exhibits analgesic and antipyretic activity. It does not possess anti-inflammatory activity. (Par) is thought to produce analgesia through a central inhibition of prostaglandin synthesis².

Codeine phosphate:

7,8-Didehydro-4,5α-epoxy-3-methoxy-17-methylmorphinan-6α-ol phosphate hemihydrate or Codeine phosphate (Cod) is a White or almost white, crystalline powder or small, colorless crystals.

Freely soluble in water, slightly soluble or very slightly soluble in ethanol(96 percent)¹, fig. 1,b.

(Cod) belongs to a group of medicines called analgesics, which are used to block pain. It is an opioid analgesic, and it acts by blocking pain and emotional response to pain². (Par) and (Cod) have been determined by various instrumental methods such as spectrophotometry^3,4,5, high performance liquid chromatography (HPLC)^6,7,8, micellar liquid chromatography⁹, electrochemical analysis^{10,11,12,13,14}.

Fig.1: Structure of the analytes:

(a) Paracetamol (Par), (b) Codeine Phosphate (Cod).

MATERIALS AND METHODS:

Apparatus:

HPLC analysis was performed on an YL 9100 HPLC system equipped with binary pump YL9111, vacuum degasser series YL9101, YL9130 column compartment and YL9120, UV/Vis. Detector (Korea). Chromatographic separations were obtained by using Shim-pack clc-C₈ column (25 cm × 4.6 mm i.d., 5 µm) (Shimadzu, Japan). Ultrasonic bath (Daihan, USA), analytical balance TE64 Sartorius (Germany) sensitivity 0.01 mg. Germany digital pipettes (Isolab).

Chemicals:

Solvents and materials were used as analytical grade: water, ethanol (Merck, Germany), acetonitrile (Isolab, Germany), all were HPLC grade. Phosphoric acid (Isolab, Germany), and methyl paraben (MP), purity 99.38 %, (Clariant, UK), (Par) purity 99.20%, (Abbott health care, India), (Cod) purity 99.60%, (Hebei Jiheng, China).

Stock standard preparation:

2.5 mg/mL of (Par) was prepared as stock standard solution by dissolving an appropriate weight of this material in hot dual distillation water, by taking the purity of the material on consideration.

2.5 mg/mL of (Cod) was prepared as stock standard solution by dissolving an appropriate weight of this material in dual distillation water, by taking the purity of the material on consideration.

2.5 mg/mL of (MP) was prepared as stock standard solution by dissolving an appropriate weight of this material in ethanol, by taking the purity of the material on consideration.

Calibration Curve:

To construct the calibration curve, seven standard solutions (3, 25, 100, 300, 400, 500, 600) 𝜇g/mL for (Par) and (1, 25, 100, 200, 400, 600, 800) 𝜇g/mL for (Cod) were prepared and the area of peaks was measured of each solution five times.

Samples preparation:

Four Syrian products were studied:

Ten "Cetacodeine" tablets were weighed and finely powdered and an accurate weight equivalent to one tablet 500 mg (Par) and 10 mg (Cod) was accurately weighed, then dissolved in 100 mL volumetric flask with hot dual distillation water. The sample solution was filtered through a filter (Whatman 3, England). Then 0.1 mL was taken to 10 mL volumetric flask to determine (Par, a) or 5 mL was taken to 10 mL volumetric flask to determine (Cod, b) and adjusted to volume with mobile phase and filtered through a 0.45 µm nylon syringe filter and degassed by ultrasonication, then 20 μL of the sample was injected into the chromatograph.

Ten "Paradrin" tablets were weighed and finely powdered and an accurate weight equivalent to one tablet 500 mg (Par) and 8 mg (Cod) was accurately weighed, then dissolved in 100 mL volumetric flask with hot dual distillation water. The sample solution was filtered through a filter (Whatman 3, England). Then 0.1 mL was taken to 10 mL volumetric flask to determine (Par, a) or 5 mL was taken to 10 mL volumetric flask to determine (Cod, b) and adjusted to volume with mobile phase and filtered through a 0.45 µm nylon syringe filter and degassed by ultrasonication, then 20 μL of the sample was injected into the chromatograph.

Ten "Cetamol Barakat" tablets were weighed and finely powdered and an accurate weight equivalent to one tablet 1000 mg (Par) was accurately weighed, then dissolved in 100 mL volumetric flask with hot dual distillation. The sample solution was filtered through a filter (Whatman 3, England). Then 0.25 mL was taken to 50 mL volumetric flask and adjusted to volume with mobile phase and filtered through a 0.45 µm nylon syringe filter and degassed by ultrasonication, then 20 μL of the sample was injected into the chromatograph.

A 5 mL of "Paradrin" syrup 160 mg/5 ml (Par) was transferred to a 25 mL volumetric flask the volume was completed to 25 mL by dual distillation water then 0.1 mL was taken to 10 mL volumetric flask and adjusted to volume with mobile phase and filtered through a 0.45 µm nylon syringe filter and degassed by ultrasonication and then the sample was injected into the chromatograph.

RESULTS AND DISCUSSION:

One of the Chromatograms of seven different standard concentrations mixtures (Par), (Cod) and (MP) as internal standard is presented in Fig. 2. Each concentration was injected five times under optimized method conditions. The chromatogram showed that (Par) was well separated from (Cod) with a good resolution and the time of analysis was achieved in less than 8 min.

Fig.2: Chromatogram of standard solutions mixture: 1- (Par) 50 µg/mL, 2- (Cod) 50 µg/mL, 3- (MP) 125 µg/mL.

Chromatographic conditions: C₈ column; mobile phase: acetonitrile: phosphoric acid (pH = 2.8) 35:65 (v/v); flow rate 1.0 mL/min, temperature 25 °C and detection at 212 nm.

Optimization of the HPLC conditions:

Mobile phase effect:

The effect of composition of the mobile phase (using C₈ column 25 cm × 4.6 mm i.d., 5 µm) on the retention time of (Par), (Cod) and the internal standard (MP) was investigated in Fig. 3. An increase in the percentage of acetonitrile decreases the retention of (Par) where as the retention of (Cod) increases. So, the optimum acetonitrile percentage to obtain a good separation was found to be 35 %.

Fig. 3: Mobile phase effect on the retention time of: (Par) 50 µg/mL, (MP) 50 µg/mL and (Cod) 50 µg/mL.

Chromatographic conditions: C₈ column; mobile phase: variable ratio of acetonitrile: phosphoric acid (pH = 2.8), flow rate 1.0 mL/min, temperature 25 °C and detection at 212 nm.

pH effect of mobile phase:

Effect of pH in the chromatographic elution of both compounds (Par) and (Cod) was investigated by changing the pH values of the aqueous component of the mobile phase from (2.5 to 4.5), fig.4. It was observed that the pH of mobile phase values from (2.5 to 3) permitted the elution of drugs in the following order (Par), (Cod) and (MP), but when the pH values became from (3 to 4.5) the order was (Par), (MP) and (Cod). pH = 2.8 was chosen for the optimum separation of these compounds.

Fig.4: pH effect of mobile phase on the retention time (t_R) of (Par) 50 µg/mL, (MP) 50 µg/mL and (Cod) 50 µg/mL.

Chromatographic conditions: C₈ column; mobile phase: acetonitrile: phosphoric acid (variable value of pH) 35:65 (v/v);

flow rate 1.0 mL/min, temperature 25 °C and detection at 212 nm.

Flow rate effect:

The optimum flow rate was identified by changing flow rate from (0.8 to 1.3) mL/min. It was concluded that each two peaks were completely separated, with a fine and symmetrical aspect at flow rate (1 mL/min) which also corresponds to the deviation point as seen in fig. 5.

Fig.5: Flow rate effect of mobile phase on the retention time of (Par) 50 µg/mL, (MP) 50 µg/mL and (Cod) 50 µg/mL.

Chromatographic conditions: C₈ column; mobile phase: acetonitrile: phosphoric acid (pH = 2.8) 35:65 (v/v);

flow rate (variable value), temperature 25 °C and detection at 212 nm.

Chromatographic conditions:

Table 1 presents the chromatographic method conditions, which were applied for simultaneous determination of (Par) and (Cod) in presence an internal standard (MP).

Table 1: (Par) and (Cod) separation chromatographic conditions, in presence an internal standard (MP).

*HPLC method*	*Specification*
Shim-pack clc-C₈(25 cm × 4.6 mm, 5 μm)	Column
Phosphoric acid (pH = 2.8): acetonitrile 65:35 (v/v),	Mobile phase
MP	Internal standard
1 mL/min	Flow rate
25 °C	Temperature
UV 212 nm	Detector
20 µL	Injection volume

METHODS VALIDATION:

Linearity:

System linearity was determined by analysis of five replicates of seven standards concentrations of (Par) and (Cod) as seen in fig. 6, 7.

Linear relationship was obtained between the peak area ratio of (Par) or (Cod) to the internal standard (MP) in function to (Par) or (Cod) concentrations.

Fig.6: Linear relations for (Par):

C₁: 3µg/mL, C_2:25: µg/mL, C₃: 100 µg/mL

C₄:300 µg/mL, C₅:400 µg/mL, C₆: 500 µg/mL

C₇: µg/mL

N = 5 for each concentration

Accuracy/recovery:

The accuracy of the method was checked by evaluating the experimental concentration of the solutions prepared for the linearity test versus the nominal concentration. Good recovery of )Par( and )Cod ( was observed as shown in table 2.

Precision:

System precision:

Five replicates (n = 5) of a standard mixture solution 0.1 mg/mL )Par( and 0.05 mg/mL )Cod) were analyzed to assess system precision. The RSD % of peak area response in Table 2 showed the satisfactory repeatability of the system (< 2 %).

Method precision:

Five replicates (n = 5) of sample solutions were analyzed in the same day to determine method precision. The low RSD % (< 2 %) showed the suitability of the method for the determination of (Par) and (Cod) in pharmaceutical formulation. The method precision was summarized in table 3.

Limit of detection (LOD) and Limit of quantification (LOQ):

The (LOD) and (LOQ) were obtained from the calibration curves. The (LOD) and (LOQ) were calculated based on the standard deviation of the intercept (SD = δ) and the slope (S) of the calibration curves using the formulae 3.3 δ/S and 10 δ/S respectively. The (LOD) and (LOQ) concentrations were reported in table 2.

Specificity:

It was determined (Par) and (Cod) in all samples without any interference. Peaks were completely separated with good resolution and specificity in all studied products. Confirming the specificity of the method where the peaks of (Par) and (Cod) in the samples were identified by comparing the retention time with that of the standards.

Application of the proposed method for estimation (Par) and (Cod) in Syrian pharmaceutical formulations:

The applicability of HPLC-UV method was investigated for pharmaceutical preparations figs. (8 - 11). In all the preparations, The amount of (Par) and (Cod) was obtained by direct measurement using the linear relationship curve. The total analysis time was less than 8 min with good resolution, good peak shapes and minimal tailing. Four pharmaceutical formulations containing (Par), (Cod) or (Par) and (Cod) were analyzed. figs. (8- a,b), (9- a,b) illustrates typical chromatograms of (Par) and (Cod) in ''Cetacodeine'' and ''Paradrin Plus'' tablets product respectively. fig. 10 illustrates a typical chromatogram of (Par) in ''Paracetamol Barakat'' tablets product. fig. 11 illustrates a typical chromatogram of (Par) in ''Paradrin'' syrup product. The dosages of (Par) and (Cod) were conformed to USP legislation¹.

Fig. 8 : Chromatogram of “Cetacodeine” tablets (a, b): 1-(Par), 2- (Cod) , 3- (MP) as internal standard 125 µg/mL.

Chromatographic conditions: C₈ column; mobile phase: acetonitrile: phosphoric acid (pH = 2.8) 35:65 (v/v); flow rate 1.0 mL/min, temperature 25 °C and detection at 212 nm.

CONCLUSION:

Proposed HPLC method is a direct, specific, accurate and precise for simultaneous determination of paracetamol and codeine phosphate in pharmaceutical formulations. The levels of pharmaceutical compounds were within the permissible limits set by the USP legislation¹. The presence of active substances in actual quantities in pharmaceutical products was conformed to the paracetamol and codeine phosphate formulations. The described method is suitable for routine analysis and quality control of pharmaceutical preparation containing these two components, either alone or in combination.

ACKNOWLEDGEMENT:

The Ministry of High Education in Syria financially and technically supported this work through department of Chemistry, Faculty of Science, University of Aleppo, Syria.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

REFERENCES:

1. United State Pharmacopeia 34. 2011.

2. Martindal the Complete Drug Reference 35. 2007.

3. Thuy DT, Hoang VD. Simultaneous Determination of Paracetamol and Codeine Phosphate In Combined Tablets by First-Order Derivative and Ratio Spectra First-Order Derivative UV Spectrophotometry. Asian J. Research Chem. 2009; 2(2): 143-147.

4. Elsayed MA, Belal SF, Elwalily AM, Abdine H. Spectrophotometric Determination of Acetaminophen, Salicylamide and Codeine Phosphate in Tablets. Analyst Journal. 1979; (104): 620-625.

5. Dinç E, Baleanu D, Onur F. Simultaneous Spectrophotometric Analysis of Codeine Phosphate Acetylsalicylic Acid and Caffeine in Tablets by Inverse Leastsquares and Principal Component Regression Techniques. Analytical Letters. 2014; 35(3): 545-558.

6. Vojta J, Hanzlík P, Jedliˇcka A, Coufal P . Separation and Determination of Impurities in Paracetamol, Codeineand Pitophenone in The Presence of Fenpiverinium in Combined Suppository Dosage Form. Journal of Pharmaceutical and Biomedical Analysis. 2015;102: 85-92.

7. Schmidt AH. Validated HPLC Method for the Determination of Residues of Acetaminophen, Caffeine, and Codeine Phosphate on Swabs Collected from Pharmaceutical Manufacturing Equipment in Support of Cleaning Validation. Journal of Liquid Chromatography & Related Technologies. 2006; 29(11): 1663-1673.

8. Maslarska V, Tencheva J. Simultaneous Determination and Validation of Paracetamol and Codeine Phosphate in Pharmaceutical Preparation by RP-HPLC. International Journal of Pharmacy and Pharmaceutical Sciences. 2013; 5(2): 417-419.

9. Belal F, Omar MA, Derayea S, Zayed S, Hammad MA, Saleh SF. Simultaneous Determination of Paracetamol, Caffeine and Codeine in Tablets and Human Plasma by Micellar Liquid Chromatography. European Journal of Chemistry. 2015; 6(4): 468-474.

10. Garazhian E, Shishehbore MR. A New Sensitive Sensor for Simultaneous Differential Pulse Voltammetric Determination of Codeine and Acetaminophen Using a Hydroquinone Derivative and Multiwall Carbon Nanotubes Carbon Paste Electrode. International Journal of Analytical Chemistry. 2015.

11. Santos AM, Silva TA, Vicentini FC, Fatibello O. Flow Injection Analysis System with Electrochemical Detection for The Simultaneous Determination of Nanomolar Levels of Acetaminophen and Codeine. Arabian J. of Chemistry. 2017.

12. Santos AM, Vicentini FC, Deroco PB, Rocha RC, Fatibello O. Square-Wave Voltammetric Determination of Paracetamol and Codeine in Pharmaceutical and Human Body Fluid Samples Using a Cathodically Pretreated Boron-Doped Diamond Electrode. J. Braz. Chem. Soc. 2015; 10(26).

13. Deroco PB, Vicentini FC, Fatibello O. An Electrochemical Sensor for the Simultaneous Determination of Paracetamol and Codeine Using a Glassy Carbon Electrode Modified with Nickel Oxide Nanoparticles and Carbon Black. Electroanalysis. 2015; 27: 2214-2220.

14. Hasanpour F, Taei M, Tahmasebi S. Ultra-Sensitive Electrochemical Sensing of Acetaminophen and Codeine in Biological Fluids Using Cuo/CuFe₂O₄ Nanoparticles as a Novel Electrocatalyst. J.of Food and Drug analysis. 2017.

Received on 02.11.2018 Modified on 16.12.2018

Research J. Pharm. and Tech. 2019; 12(3): 1327-1332.

DOI: 10.5958/0974-360X.2019.00222.1

Product	Company	Pharmaceutical compounds	(mg/dose)	Per %	RSD %	Rec (%)
Cetacodeine (tablets)	Ultra Medica	(Par) 500 mg/tab	501.85	100.37	0.85	101.24
Cetacodeine (tablets)	Ultra Medica	(Cod) 10 mg/tab	9.85	98.50	1.62	100.78
Paradrin Plus (tablets)	Avenzor	(Par) 500 mg/tab	503.90	100.78	1.02	100.31
Paradrin Plus (tablets)	Avenzor	(Cod) 8 mg/tab	8.12	101.50	1.77	99.97
Paracetamol Barakat (tablets)	Barakat	(Par) 1000 mg/tab	1003.60	100.36	0.91	101.05
Paradrin (syrup)	Avenzor	(Par) 160 mg/5 ml	163.74	102.34	1.03	99.89