Piroxicam Capsules-Assay and Organic Impurities Following United States Pharmacopoeia Pending Forum Method

Dr Sanjay Poman,
Mumbai Application Laboratory
India

Introduction

Piroxicam (Figure 1) is an oxicam NSAID (non-steroidal anti-inflammatory drug) and is used to relieve the symptoms of painful inflammatory conditions like arthritis.¹

A simple, precise, and sensitive reverse-phase high-performance liquid chromatography (RP-HPLC) gradient method (Table 1) using an Ascentis^® Express C18 column was adopted for the assay and organic impurities analysis of Piroxicam capsules as part of method modernization from the USP-NF.² The method was assessed in relation to USP general chapters <621>, <1225>, and <1226>.

Figure 1. Chemical structure of Piroxicam

Piroxicam Capsules Assay And System Suitability Test

Table 1.Chromatographic conditions

Experimental
Experimental Conditions
Column:	Ascentis®Express C18 100 x 4.6 mm I.D., 2.7µm (53827-U)
Mobile Phase:	[A] Formic acid: ammonium hydroxide: water (0.1:0.35:100 v/v/v); [B] Acetonitrile
Gradient:	Time A (%) B (%)    0.0 85 15    2.0 85 15    5.0 40 60    9.0 40 60  12.0 60 40  12.1 60 40  15.0 85 15
Flow rate:	1 mL/min
Pressure drop:	167 bar (2421 psi)
Column temp.:	30° C
Autosampler temp:	4° C
Detectior:	UV @ 254 nm (analytical flow cell, 10 µL)
Injection:	5 µL
Samples
System suitability solution:	Organic Impurities: 1.0 mg/mL of Piroxicam RS and 10 μg/mL Piroxicam Related Compound B RS mobile phase
Standard solutions:	Assay: Dissolve 0.1 mg/mL of Piroxicam RS in methanol Organic Impurities: 2 μg/mL of Piroxicam RS and 10 μg/mL of Piroxicam Related Compound A in methanol
Sensitivity solution:	Organic impurities: 0.5 μg/mL of Piroxicam RS in methanol, sonicate if needed.
Sample solutions:	Assay (0.1 mg/mL): Transfer a portion of the mixed content of capsules equivalent to 10 mg of piroxicam to a 100 mL volumetric flask, add about 80 ml of methanol, and swirl to disintegrate. Sonicate for 5 minutes and agitate by mechanical means for 30 minutes. Dilute with methanol to volume. Centrifuge to obtain a clear solution. Organic impurities (1 mg/mL): Transfer a portion of the mixed content of capsules equivalent to 20 mg of piroxicam in a 20 mL volumetric flask. Add 15 mL of methanol, and swirl to disintegrate. Sonicate for 5 minutes and agitate by mechanical means for 30 minutes. Dilute with methanol to volume. Centrifuge to obtain a clear solution.

Results

Assay

Figure 2. Chromatographic blank run.

Figure 3. Chromatogram of a piroxicam standard solution (0.1 mg/mL).

Chromatographic Data for Standard Solution
Peak	Compound	Retention Time (min)	Tailing Factor	Area
1	Piroxicam	4.1	1.5	952666

Figure 4. Chromatogram of piroxicam sample solution (0.1 mg/mL)

Chromatographic Data for Sample Solution
Peak	Compound	Retention Time (min)	Tailing Factor	Area
1	Piroxicam	4.2	1.4	950353

Table 2.Repeatability Standard Solution 0.1 mg/mL piroxicam

Sample (0.1 mg/mL)	Area
STD 1	952621
STD 2	938699
STD 3	946489
STD 4	947663
STD 5	952666
Mean	947627.6
Standard Deviation	5125.17
RSD (%)	0.54

Table 3.System Suitability - USP Specification and observed values for piroxicam Standard Solution (0.1 mg/mL) using the Ascentis^® Express C18 column

System Suitability Requirements
Component	USP Specification	Observed value
% RSD	NMT 2.5%	0.54%
Tailing Factor	NMT 2.0	1.5

Organic Impurities Analysis of Piroxicam Capsules

Figure 5. Chromatogram of the piroxicam sensitivity solution (0.5 µg/mL).

Chromatographic Data - Sensitivity Solution
Peak	Compound	Retention Time (min)	Tailing Factor	Area	S/N	USP Requirement S/N
1	Piroxicam 0.5 µg/ml	4.1	1.02	16729	15.2	NLT 10

Figure 6. Chromatogram standard solution (2 µg/mL piroxicam and impurity A at 10 µg/mL).

Chromatographic Data – System Suitability Solution
Peak	Compound	Retention Time (min)	RRT	Tailing Factor	Resolution	Area
1	Impurity A	1.7	0.41	1.02	-	13705
2	Piroxicam	4.1	1	1.36	31.2	19639

Figure 7. Chromatographic separation of standard solution organic impurities with 1 mg/mL piroxicam and impurity B at 10 µg/mL.

Chromatographic Data - Standard Solution Organic Impurities
Peak	Compound	Retention Time (min)	RRT	Tailing Factor	Resolution	Area
1	Piroxicam	3.9	-	2.7	-	11088453
2	Impurity B	5.2	1.33	1.0	8.8	1115

Table 4.System Suitability Organic Impurities* - USP Specification and observed values for the using the Ascentis^® Express C18 column
* %RSD for piroxicam and piroxicam related compound A standard solution was not determined.

System Suitability Requirements*
Component	USP Specification	Observed value
RRT Impurity A	0.35	0.4
RRT Impurity B	1.1	1.3
Resolution between Piroxicam & Impurity B	NLT 4.0	8.8

Figure 8. Calibration curve for piroxicam.

Table 5.Calibration Data - Piroxicam

Concentration (μg/mL)	Mean Area
0.05	1215
0.5	12157
1	24314
10	75328
25	256425
50	509595
80	786270
100	982838
120	1179405
STEYEX	12771
Slope	9856
LOD (μg/mL)	4.27
LOQ (μg/mL)	12.9

Figure 9. Calibration curve for impurity A.

Table 6.Calibration Data- Piroxicam Impurity A

Concentration (μg/mL)	Mean Area
0.005	100
0.05	1002
0.5	2002
1	20098
2.5	50245
5	100490
8	160784
10	200960
12	241152
STEYEX	2725
Slope	20085
LOD (μg/mL)	0.4
LOQ (μg/mL)	1.3

Figure 10. Calibration curve for impurity B.

Table 7.Calibration Data- Piroxicam Impurity B

Concentration (μg/mL)	Mean Area
0.005	60
0.05	111
0.5	657
1	1311
2.5	3280
5	6576
8	10960
10	13152
12	15782
STEYX	150.3
Slope	1325
LOD (μg/mL)	0.37
LOQ (μg/mL)	1.1

Conclusion

A sensitive, stability indicating gradient reversed-phase HPLC method has been developed for the quantitative estimation of piroxicam (assay) and organic impurities in a dosage form. System suitability criteria mentioned in the USP monograph for reference standard as well as the test solution of piroxicam dosage form were found to be in compliance with % RSD and tailing factor parameters for the assay. The developed method was found to be linear for piroxicam up to 120 µg/mL with LOD of 4.2 µg/mL and LOQ of 12.9 µg/mL. For the organic impurity A linearity was assessed up to 12 µg/mL with LOD and LOQ at 0.4 and 1.3 µg/mL respectively, for impurity B also up to 12 µg/mL with LOD and LOQ at 0.37 and 1.1 µg/mL respectively. The relative retention times for impurities A and B are in agreement with the RRT values mentioned in the USP monograph. %RSD for piroxicam and piroxicam related compound A standard solution was not determined. The method can be applied for related substances study of piroxicam dosage form using the Ascentis^® Express C18 HPLC column.

See other technical articles regarding USP methods.

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References

1. Piroxicam (oral route). [Internet]. Mayocline.org:[updated 05 Oct 2023; cited 16 Oct 2023]. Available from: https://www.mayoclinic.org/drus-supplements/piroxicam-oral-route/description/drg-20069771

2. United States Pharmacopeia (2022). USP Monographs, Piroxicam.(USP-NF. Rockville, MD: United States Pharmacopeia). https://doi.org/10.31003/uspnf_m65720_05_01