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HomeSmall Molecules Analysis & Quality Control (QC)Piroxicam Capsules-Assay and Organic Impurities Following United States Pharmacopoeia Pending Forum Method

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>.

Chemical structure of Piroxicam

Figure 1.Chemical structure of Piroxicam

Piroxicam Capsules Assay And System Suitability Test

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.
Table 1.Chromatographic conditions

Results

Assay

Chromatographic blank run

Figure 2.Chromatographic blank run.

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

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

Chromatographic Data for Standard Solution

PeakCompoundRetention Time (min)Tailing FactorArea
1Piroxicam4.11.5952666
Chromatogram of piroxicam sample solution (0.1 mg/mL)

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

Chromatographic Data for Sample Solution

PeakCompoundRetention Time (min)Tailing FactorArea
1Piroxicam4.21.4950353
Sample (0.1 mg/mL)Area
STD 1952621
STD 2938699
STD 3946489
STD 4947663
STD 5952666
Mean947627.6
Standard Deviation5125.17
RSD (%)0.54
Table 2.Repeatability Standard Solution 0.1 mg/mL piroxicam

System Suitability Requirements

ComponentUSP SpecificationObserved value
% RSDNMT 2.5%0.54%
Tailing FactorNMT 2.01.5
Table 3.System Suitability - USP Specification and observed values for piroxicam Standard Solution (0.1 mg/mL) using the Ascentis® Express C18 column

Organic Impurities Analysis of Piroxicam Capsules

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

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

Chromatographic Data - Sensitivity Solution

PeakCompoundRetention Time (min)Tailing FactorAreaS/NUSP Requirement S/N
1Piroxicam 0.5 µg/ml4.11.021672915.2NLT 10
Chromatogram standard solution (2 µg/mL piroxicam and impurity A at 10 µg/mL).

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

Chromatographic Data – System Suitability Solution

PeakCompoundRetention Time (min)RRTTailing FactorResolutionArea
1Impurity A1.70.411.02-13705
2Piroxicam4.111.3631.219639
Chromatographic separation of standard solution organic impurities with piroxicam and impurity B

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

PeakCompoundRetention Time (min)RRTTailing FactorResolutionArea
1Piroxicam3.9-2.7-11088453
2Impurity B5.21.331.08.81115

System Suitability Requirements*

ComponentUSP SpecificationObserved value
RRT Impurity A0.350.4
RRT Impurity B1.11.3
Resolution between Piroxicam & Impurity BNLT 4.08.8
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.
Calibration curve for piroxicam.

Figure 8.Calibration curve for 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
Table 5.Calibration Data - Piroxicam
Calibration curve for impurity A.

Figure 9.Calibration curve for 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
Table 6.Calibration Data- Piroxicam Impurity A
Calibration curve for impurity B.

Figure 10.Calibration curve for 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
Table 7.Calibration Data- Piroxicam Impurity B

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
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