It is important to follow appropriate safety precautions when handling these drugs and to adhere to the manufacturer’s instructions for the assay procedure of SOP for Amlodipine 5mg/Valsartan 160mg/Hydrochlorothiazide 12.5mg Tablet Assay Testing.
PURPOSE:
To describe the procedure for analysis at the in-process and finished stage of SOP for Amlodipine 5mg/Valsartan 160mg/Hydrochlorothiazide 12.5mg Tablet Assay Testing.
SCOPE:
This SAP gives a detailed outline for the finished product analysis of SOP for Amlodipine 5mg/Valsartan 160mg/Hydrochlorothiazide 12.5mg Tablet Assay Testing and will cover In process testing activities on physical, chemical & instrumental basis.
RESPONSIBILITY:
QC Analyst is responsible for physical / chemical testing and preparing standard analytical procedure.
It is the responsibility of QC Manager to assist and ensure Testing Procedure as per SAP and to make certain that this SAP is followed in its entirety, reviewed regularly and revised as necessary.
REFERENCE:
USP 43, NF 38
MATERIAL AND EQIUPMENT:
- HPLC
- Dissolution apparatus
- Friability apparatus
- Hardness apparatus
- Disintegration Tester
- Vernier Caliper
- Analytical Balance
- pH meter
- Moisture analyzer
- Mortar and pestle
- Spatula
- Filter Paper
- Magnetic Stirrer & Hot Plate
- Sonicator
- Vacuum Pump
- Glassware Ammonium Acetate
- Triethylamine
- Acetonitrile HPLC Grade
- Methanol HPLC Grade
- Distilled water
PROCEDURE:
FINAL MIX
SOP for Amlodipine 5mg/Valsartan 160mg/Hydrochlorothiazide 12.5mg Tablet Assay Testing
Sample preparation: Weigh and finely powder 20 tablets of Amlodipine, Valsartan, and Hydrochlorothiazide. Mix the powders thoroughly to ensure a homogeneous blend.
Mobile phase preparation: Prepare a mobile phase by mixing 900 mL of water with 100 mL of methanol.
Chromatographic conditions: Use a reverse-phase high-performance liquid chromatography (HPLC) with a C18 column (250 mm x 4.6 mm, 5 µm particle size) and a UV detector set at 254 nm.
Calibration standard preparation: Prepare a stock solution of Amlodipine, Valsartan, and Hydrochlorothiazide by dissolving each drug separately in methanol to make a concentration of 1 mg/mL. Dilute the stock solutions to make three separate working solutions of 10 µg/mL for Amlodipine, 20 µg/mL for Valsartan, and 25 µg/mL for Hydrochlorothiazide.
Chromatographic procedure: Inject 20 µL of the sample solution and each calibration standard into the HPLC system. Use the mobile phase to elute the drugs from the column. Measure the peak areas of each drug in the calibration standards and the sample solution.
Calculation: Calculate the amount of Amlodipine, Valsartan, and Hydrochlorothiazide in the sample solution using the peak area ratios of the drugs in the calibration standards. Compare the results to the label claim for each drug in the tablet formulation.
Description:
white granular powder.
Procedure: Take 2.0 g of the test sample in a watch glass and observe visually with black background. Check the appearance of color, nature and any visible foreign particles.
Identification: (By HPLC)
A- The UV absorption spectra of the amlodipine, valsartan, and hydrochlorothiazide peaks of Sample solution A, Sample solution B, and Sample solution C, and those of the Standard solution exhibit maxima and minima at the same wavelengths, as obtained in the Assay.
B- The retention times of the amlodipine, valsartan, and hydrochlorothiazide peaks of Sample solution, Sample solution B, and Sample solution Correspond to those of the Standard solution, as obtained in the Assay.
Loss on Drying: (By Moisture Analyzer)
Procedure:
Turn on instrument 20 minute before the test. Take 2 g powder spread uniformly on Moisture Analyzer plate. Close the lid and press start button.
NMT 3.0% when determined by LOD Apparatus, Use 2.0 g of powder.
Assay: (Limit: 92.5 %-107.5%)Compression Weight of Powder/Tablet:
Amlodipine, Valsartan, and Hydrochlorothiazide Tablets contain NLT 92.5% and NMT 107.5% each of the labeled amounts of amlodipine (C20H25CIN2O5), valsartan (C24H29N5O3), and hydrochlorothiazide (C7H8CIN3O4S2)
Procedure:
Use amber glassware for all solutions containing drug substances.
Solution A: Acetonitrile, water, and phosphoric acid(50:950:1)
Solution B: Acetonitrile, water, and phosphoric acid(950:50:1)
Mobile phase: See Table 1.
Mobile Phase:
| Time (Minutes） | Solution A (%) | Solution B (%) |
| 0 | 95 | 5 |
| 6 | 50 | 50 |
| 7 | 40 | 60 |
| 10 | 50 | 50 |
| 11 | 95 | 5 |
| 15 | 95 | 5 |
Diluent: Acetonitrile and water (500:500)
0.1% Phosphoric acid: Water and phosphoric acid(1000:1)
Standard solution: 0.14 mg/mL of USP Amlodipine Besylate RS, 0.064 mg/mL of USP Valsartan RS, and 0.025mg/mL of USP Hydrochlorothiazide RS in Diluent
Sample stock solution: Transfer NLT 10 Tablets into a suitable volumetric flask. Add 0.1% Phosphoric acid to 4%of the total volume to disperse the Tablets. Sonicate for 10min. Add4% of the total volume of acetonitrile, swirl to mix, and add 60% of the total volume of Diluent. Sonicate for 20 min. Dilute with Diluent to volume to obtain solutions of nominal concentrations stated in Table 2. Centrifuge, and use the clear supernatant.
Table 2
| Tablet Strength Amlodipine / Valsartan / Hydrochlorothiazide (mg/mg/mg) | Nominal Concentration of Amlodipine (mg/mL) | Nominal Concentration of Valsartan (mg/mL) | Nominal Concentration of Hydrochlorothiazide (mg/mL) |
| 5/160/12.5 | 0.1 | 3.2 | |
| 10/160/12.5 | 0.2 | 3.2 | |
| 5/160/25 | 0.1 | 3.2 | |
| 10/160/25 | 0.2 | 3.2 | |
| 10/320/25 | 0.1 | 3.2 |
Sample solution A: Nominally equivalent to 0.1 mg/mL of amlodipine in Diluent from Sample stock solution
Sample solution B: Nominally equivalent to 0.064 mg/mL of valsartan in Diluent from Sample stock solution
Sample solution C: Nominally equivalent to 0.025 mg/mL of hydrochlorothiazide in Diluent from Sample stock solution
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 225 nm. For Identification A, use a diodearray detector in the range of 200-400 nm.
Column: 4.6-mm x 15-cm; 3-µm packing L1
Column temperature: 40°
Flow rate: 1.5 mL/min
Injection volume: 10 µL
System suitability
Sample: Standard solution
Suitability requirements
Tailing factor: NMT 2.0 for amlodipine, valsartan, and hydrochlorothiazide
Relative standard deviation: NMT 2.0% for amlodipine, valsartan, and hydrochlorothiazide
Analysis
Samples: Standard solution, Sample solution A, Sample solution B, and Sample solution C
Calculate the percentage of the labeled amount of amlodipine (C20H25CIN2O5) in the portion of Tablets taken:
Result =(Ru/Rs) x (Cs/Cu) x (M r1/M r2) X 100
Ru=peak response of amlodipine from Sample solution A
Rs =peak response of amlodipine from the Standard solution
Cs =concentration of USP Amlodipine Besylate RS in the Standard solution (mg/mL)
C u =nominal concentration of amlodipine in Sample solution A (mg/mL)
Mrl=molecular weight of amlodipine, 408.88
M r2 =molecular weight of amlodipine besylate, 567.05
Calculate the percentage of the labeled amount of valsartan(C24H29N5O3) in the portion of Tablets taken: Result =(Ru/Rs) x (Cs/Cu) x 100
Ru =peak response of valsartan from Sample solution B
Rs =peak response of valsartan from the Standard solution
Cs = concentration of USP Valsartan RS in the Standard solution (mg/mL)
C u =nominal concentration of valsartan in Sample solution B(mg/mL)
Calculate the percentage of the labeled amount of hydrochlorothiazide (C7H8CIN3O4S2) in the portion of Tablets taken:
Result =(Ru/Rs) x (Cs/Cu) x 100
Ru=peak response of hydrochlorothiazide from Sample solution C
Rs=peak response of hydrochlorothiazide from the Standard solution
C s =concentration of USP Hydrochlorothiazide RS in the Standard solution (mg/mL)
C u =nominal concentration of hydrochlorothiazide in Sample solution C (mg/mL)
Acceptance criteria: 92.5%-107.5%
Calculations:
Average Sample AUC x Standard Concentration x Ave. Weight of Tablet x Potency
Average Standard AUC x Sample Concentration x Label Claim x Factor
(Limit: for Amlodipine Valsartan and Hydrochlorothiazide:92.5%–107.5%of the labeled amount)
Uniformity of Dosage Units: (By Weight Variation)
Weigh 20 tablets individually and calculate the average weight as:
Average weight = (Weight Of 20 Tablets)/20
Weigh all these tablets individually and mark among these weights maximum (WMax) & minimum (WMin) weight tablets. Calculate the maximum and minimum variation in percent as:
Maximum% age variation =((WMax) – (WAvg.))/WAvg×100
Minimum % age variation ((WMin) – (WAvg.))/WAvg×100
Standard Limit: ± 5 %
Hardness Test & Dimensions:
Perform the hardness test on 10 tablets and take the average. Power on the instrument and regulate zero adjustment and preset the ‘’HARDNESS’’ and “Thickness” mode. Place the tablet on the test plate, begin testing and read the hardness thickness and diameter. Clean the shattered tablet and print out test results, perform the test on 10 tablets and take the average.
Hardness Standard Limit: Not more than 15.00 Kg
Friability Test:
For tablets with a unit mass equal to or less than 650 mg, take a sample of whole tablets corresponding to 6.5 g. For tablets with a unit mass of more than 650 mg, take a sample of 10 whole tablets. The tablets should be carefully dedusted prior to testing. Accurately weigh the tablet sample, and place the tablets in the drum. Rotate the drum 100 times, and remove the tablets. Remove any loose dust from the tablets as before, and accurately weigh. Generally, the test is run once. If obviously cracked, cleaved, or broken tablets are present in the tablet sample after tumbling, the sample fails the test. If the results are doubtful or if the weight loss is greater than the targeted value, the test should be repeated twice and the mean of the three tests determined. A maximum mean weight loss from the three samples of not more than 1.0% is considered acceptable for most products.
% FRIABILITY= W1-W2/ W1 x 100
Disintegration Test:
Place the beaker filled with water into the beaker stand inside the bath and. Switch on the instrument, set the temperature at 37ºC± 2º C and wait till the temperature of the beaker reaches to the 37ºC± 2º C. Engage the basket on basket hook put the tablets in 6 tubes individually and place the discs then start the oscillations and timer. Note the disintegration time of all the six tablets.
Note the Disintegration time of 1st tablet to the last tablet.
Disintegration Time: NMT 15 minutes at 370C ± 20C
Acceptance criteria of disintegration for tablets:
If 1 or 2 tablets fail to disintegrate completely repeat the test on 12 additional tablets, not less than 16 of the totals of 18 tablets tested disintegrate completely.
Dissolution Test:
Buffer: Dissolve 6.805 9 of monobasic potassium phosphate and 0.896 9 of sodium hydroxide in 1000 mL of water. Adjust with 0.2 N sodium hydroxide or 1 M phosphoric acid to a pH of 6.8.
USP Apparatus: USP Apparatus II
Medium: 900 mL Buffer pH 6.8 (50 mM Phosphate Buffer Solution PBS)
For 5/160/12.5, 10/160/12.5, 5/160/25, and 10/160/25 (mg/mg/mg) of Tablet strengths (amlodipine/valsartan/hydrochlorothiazide):50 rpm
For 10/320/25 (mg/mg/mg) of Tablet strengths (amlodipine/valsartan/hydrochlorothiazide):55rpm
Time: 30 min
Solution A: Acetonitrile, water, and phosphoric acid (50:950:1)
Solution B: Acetonitrile, water, and phosphoric acid (950:50:1)
Mobile phase: See Table 3.
| Time (Minutes） | Solution A (%) | Solution B (%) |
| 0 | 67 | 33 |
| 2.5 | 23 | 77 |
| 2.51 | 67 | 33 |
| 4 | 67 | 33 |
Diluent: 1 mg/mL of polysorbate 80 in Buffer
Standard stock solution A: 0.07 mg/mL of USP Amlodipine Besylate and 0.124 mg/mL of USP
Hydrochlorothiazide RS. Initially dissolve with 4% of the total volume of methanol, and dilute with Diluent to volume.
Standard stock solution B: 3.2 mg/mL of USP Valsartan RS in methanol
Standard solution: 0.014 mg/mL of USP Amlodipine Besylate RS, 0.16 mg/mL of USP Valsartan RS, and 0.0248mg/mL of USP Hydrochlorothiazide RS in Diluent from Standard stock solution A and Standard stock solution B, respectively.
Sample solution: Pass a portion of the solution under test through a suitable filter of 0,45-µm pore size. Discard at least the first 10 mL of the filtrate.
Chromatographic system
(See Chromatography (621), System Suitability.)
Mode: LC
Detector: UV 250 nm
Column: 4.6-mm x 5-cm; 3-µm packing L1
Column temperature: 30°
Flow rate: 1.5 mL/min
Injection volume
For 10/320/25 (mg/mg/mg) of Tablet strengths(amlodipine/valsartan/hydrochlorothiazide): 5 µL
For 5/160/12.5, 10/160/12.5,5/160/25, and10/160/25 (mg/mg/mg) of Tablet strengths(amlodipine/valsartan/hydrochlorothiazide):10 µL
System suitability
Sample: Standard solution
Suitability requirements
Resolution: NLT 3.0 between amlodipine and valsartan
Tailing factor: NMT2.0 for amlodipine, valsartan, and hydrochlorothiazide
Relative standard deviation: NMT2.0% for amlodipine, valsartan, and hydrochlorothiazide
Analysis
Samples: Standard solution and Sample solution
Calculate the percentage of the labeled amount of amlodipine (C20H25CIN2O5) dissolved:
Result=(Ru/Rs) x CsxVx (Mr1/Mr2) x (1/L) x 100
Ru= peak response of amlodipine from the Sample solution
Rs=peak response of amlodipine from the Standard solution
Cs=concentration of USP Amlodipine Besylate RS in the Standard solution(mg/mL)
V= volume of Medium, 900 mL
Mr1= molecular weight of amlodipine, 408.88
Mr2=molecular weight of amlodipine besylate, 567.05
L=label claim of amlodipine (mg/Tablet)
Calculate the percentage of the labeled amount of valsartan (C24H29N5O3) dissolved:
Result=(Ru/Rs) x Cs x V x (1/L) x 100
Ru = peak response of valsartan from the Sample solution
Rs = peak response of valsartan from the Standard solution
Cs = concentration of USP Valsartan RS in the Standard solution (mg/ml)
V = volume of Medium, 900 mL
L2 = label claim of valsartan (mg/Tablet)
Calculate the percentage of the labeled amount of hydrochlorothiazide (C7H8CIN3O4S2) dissolved:
Result=(Ru/Rs) x Cs x V x (1/L) x 100
Ru= peak response of hydrochlorothiazide from the Sample solution
Rs= peak response of hydrochlorothiazide from the Standard solution
Cs= concentration of USP Hydrochlorothiazide RS in the Standard solution (mg/ml)
V= volume of Medium, 900 ml
L= label claim of hydrochlorothiazide (mg/Tablet)
Tolerances: NLT 75% (Q) of the labeled amount of amlodipine (C20H25ClN2O5) is dissolved, NLT 80% (Q) of the labeled amount of valsartan (C24H29N5O3) is dissolved, and NLT 80% (Q) of the labeled amount of hydrochlorothiazide (C7H8CIN3O4S2) is dissolved.
Leak Test:
Take 06 blisters of tablet 03 filled and 03 empty. Dip these blisters in the bowl of leak test apparatus containing colored water. Create vacuum 250 mmHg and hold for one minute. Release vacuum and check the penetration of water inside the blisters. All blisters should be free of water.