Calcium and Vitamin D with Minerals Samples: Standard solution and System suitability Tablets

Transcription

1 USP 35 Dietary Supplements / Calcium C = measured concentration of calcium in the PERFORMANCE TESTS DISINTEGRATION AND DISSOLUTION OF DIETARY SUPPLEMENTS C U = nominal concentration of calcium in the 2040 : Meet the requirements for Dis with respect to calcium Acceptance criteria: 90.0% 125.0% of the labeled Medium: 0.1 N ; 900 ml amount of calcium (Ca) Apparatus 2: 75 rpm CHOLECALCIFEROL OR ERGOCALCIFEROL (VITAMIN D) Time: 30 min [NOTE Use low-actinic glassware throughout this : Determine the amount of calcium (Ca) procedure.] dissolved, using the procedure in Calcium, making any Mobile phase: n-hexane and isopropyl alcohol (99:1) necessary volumetric adjustments. Standard : 2 µg/ml of USP Ergocalciferol RS or Tolerances: NLT 75% of the labeled amount of Ca is USP Cholecalciferol RS in n-hexane dissolved. : Heat a volume of Standard WEIGHT VARIATION OF DIETARY SUPPLEMENTS 2091 : Meet at 60 for 1 h to partially isomerize vitamin D the requirements (ergocalciferol or cholecalciferol) to its corresponding precursor. CONTAMINANTS Sample : Weigh, and grind NLT 20 Tablets. MICROBIAL ENUMERATION TESTS 2021 : The total aerobic Transfer the equivalent to 20 µg of cholecalciferol or microbial count does not exceed 3000 cfu/g, and the ergocalciferol to a container having a polytef-lined screw total combined molds and yeasts count does not exceed cap. Add 8 ml of dimethyl sulfoxide and 12 ml of n- 300 cfu/g. hexane, and shake for 45 min on a wrist-action shaker MICROBIOLOGICAL PROCEDURES FOR ABSENCE OF SPECIFIED with tubes in a water bath maintained at 60. Centrifuge MICROORGANISMS 2022 : Meet the requirements of the for 10 min, withdraw the hexane layer by means of a tests for absence of Salmonella species, Escherichia coli, pipet, and transfer to an evaporation flask. Add 12 ml of and Staphylococcus aureus n-hexane to the dimethyl sulfoxide layer, mix on a vortex mixer for 5 min, and again withdraw the hexane ADDITIONAL REQUIREMENTS PACKAGING AND layer by means of a pipet, and add to the evaporation STORAGE: Preserve in tight, light-resistant flask. Repeat this extraction with three additional 12-mL containers. portions of n-hexane, adding the hexane extracts to the LABELING: The label states that the product is Calcium evaporation flask. Evaporate the combined hexane with Vitamin D Tablets. The label states also the quantities extracts in vacuum at room temperature to dryness. of calcium and Vitamin D in terms of metric units/tablet, Dissolve in and dilute the residue in a volume of n- and the salt form of calcium and the chemical form of hexane to obtain a concentration of 2 µg/ml. Vitamin D present in the Tablet. USP REFERENCE STANDARDS Chromatographic system 11 USP Cholecalciferol RS USP Ergocalciferol RS Detector: UV 265 nm Column: 4.6-mm 15-cm; 3-µm packing L8 Injection size: 100 µl Calcium and Vitamin D with Minerals Samples: Standard and Tablets Flow rate: 1 ml/min DEFINITION Re: NLT 10 between the vitamin D form Calcium and Vitamin D with Minerals Tablets contain Vitapresent and its corresponding precursor, System min D as Ergocalciferol (Vitamin D 2) or Cholecalciferol (Visuitability tamin D 3), Calcium, and one or more minerals derived Relative standard deviation: NMT 3.0%, Standard from substances generally recognized as safe, furnishing one or more of the following elements in ionizable form: copper, magnesium, manganese, and zinc. Tablets con- Samples: Standard and Sample tain NLT 90.0% and NMT 165.0% of the labeled amount Measure the peak areas for vitamin D. Calculate the of vitamin D, as cholecalciferol (C 27H 44O) or ergocalciferol percentage of the labeled amount of cholecalciferol (C 28H 44O), and NLT 90.0% and NMT 125.0% of the la- (C 27H 44O) or ergocalciferol (C 28H 44O) in the portion of beled amounts of calcium (Ca), copper (Cu), magnesium Tablets taken: (Mg), manganese (Mn), and zinc (Zn). They may contain other labeled added substances that are generally recog- Result = (r U/r S) (C S/C U) F 100 nized as safe, in amounts that are unobjectionable. r U = peak area of cholecalciferol or ergocalciferol STRENGTH from the Sample CHOLECALCIFEROL or ERGOCALCIFEROL (VITAMIN D) r S = peak area of cholecalciferol or ergocalciferol [NOTE Use low-actinic glassware throughout this from the Standard procedure.] C S = concentration of USP Cholecalciferol RS or Mobile phase: n-hexane and isopropyl alcohol (99:1) USP Ergocalciferol RS in the Standard Standard : 2 µg/ml of USP Ergocalciferol RS or (µg/ml) USP Cholecalciferol RS in n-hexane C U = nominal concentration of cholecalciferol or : Heat a volume of Standard ergocalciferol in the at 60 for 1 h to partially isomerize vitamin D F = correction factor to account for the average (ergocalciferol or cholecalciferol) to its corresponding amount of previtamin D present in the precursor. Sample, 1.09 Sample : Weigh NLT 20 Tablets, and grind the Acceptance criteria: 90.0% 165.0% of the labeled Tablets to a fine powder. Transfer the equivalent of 20 amount of vitamin D as cholecalciferol (C 27H 44O) or ergocalciferol (C 28H 44O) µg of cholecalciferol or ergocalciferol to a container hav- ing a polytef-lined screw cap. Add 8 ml of dimethyl sulfoxide and 12 ml of n-hexane, and shake for 45 min on a wrist-action shaker with tubes in a water bath main-

2 1222 Calcium / Dietary Supplements USP 35 tained at 60. Centrifuge for 10 min, withdraw the hex- Lanthanum chloride, and dilute with water to ane layer by means of a pipet, and transfer to an evapo- volume to obtain Standard s having ration flask. Add 12 ml of n-hexane to the dimethyl concentrations of 1.0, 1.5, 2.0, 2.5, and 3.0 µg/ml of sulfoxide layer, mix on a vortex mixer for 5 min, and calcium. again withdraw the hexane layer by means of a pipet, Sample stock : Weigh and finely powder NLT and add to the evaporation flask. Repeat this extraction 20 Tablets. Mix a portion of the powder equivalent to a with three additional 12-mL portions of n-hexane, add- nominal amount of 500 mg of calcium with 25 ml of ing the hexane extracts to the evaporation flask. Evapo- concentrated, and heat for 30 min on rate the combined hexane extracts in vacuum at room a steam bath. Cool, dilute with water to 1000 ml, and temperature to dryness. Dissolve in and dilute the resi- filter. due in a volume of n-hexane to obtain a concentration Sample : Quantitatively dilute a volume of the of 2 µg/ml. Sample stock with N to Chromatographic system obtain a nominal concentration of 100 µg/ml of calcium. Transfer 2.0 ml of this to a 100-mL volumetric flask, add 1.0 ml of Lanthanum chloride Detector: UV 265 nm, and dilute with water to volume. Column: 4.6-mm 15-cm; 5-µm packing L8 Flow rate: 1 ml/min (See Spectrophotometry and Light-Scattering 851.) Injection size: 100 µl Lamp: Calcium hollow-cathode Samples: Standard and Flame: Nitrous oxide acetylene Analytical wavelength: Calcium emission line, nm Re: NLT 10 between the vitamin D form Blank: N containing 1 ml of present and its corresponding precursor, System suita- Lanthanum chloride /100 ml bility Relative standard deviation: NMT 3.0%, Standard Determine the absorbances of the s against the Blank. Plot the absorbances of the Standard s Samples: Standard and Sample versus concentration, in µg/ml, of calcium, and draw Measure the responses for the vitamin D peaks. Calcu- the straight line best fitting the five plotted points. late the percentage of the labeled amount of chole- From the graph, determine the concentration, in µg/ calciferol (C 27H 44O) or ergocalciferol (C 28H 44O) in the ml, of calcium in the Sample. portion of Tablets taken: calcium (Ca) in the portion of Tablets taken: Result = (r U/r S) (C S/C U) F 100 r U = peak height for cholecalciferol or ergocalciferol from the Sample C = measured concentration of calcium in the r S = peak height for cholecalciferol or ergocalciferol from the Standard C U = nominal concentration of calcium in the C S = concentration of USP Ergocalciferol RS or USP Cholecalciferol RS in the Standard Acceptance criteria: 90.0% 125.0% of the labeled (µg/ml) amount of Calcium (Ca) C U = nominal concentration of ergocalciferol or COPPER, Method 1 cholecalciferol in the Copper standard : Dissolve 1.00 g of copper F = correction factor to account for the average foil in a minimum volume of a 50% (v/v) of amount of previtamin D present in the nitric acid, and dilute with a 1% (v/v) Sample, 1.09 of nitric acid to 1000 ml. This contains 1000 Acceptance criteria: 90.0% 165.0% of the labeled µg/ml of copper. amount of cholecalciferol (C 27H 44O) or ergocalciferol Standard stock : 100 µg/ml of copper from (C 28H 44O) Copper standard diluted with N CALCIUM, Method 1 [NOTE A commercially available atomic absorption Standard s: To separate 200-mL volumetric standard for calcium may be used where flasks transfer 1.0, 2.0, 4.0, 6.0, and 8.0 ml of the preparation of a Calcium standard stock is Standard stock. Dilute with water to volume to described in the following section. Concentrations of obtain concentrations of 0.5, 1.0, 2.0, 3.0, and 4.0 the Standard s and the Sample stock may µg/ml of copper. be modified to fit the linear or working range of the Sample : Weigh and finely powder NLT 20 instrument.] Tablets. Transfer the equivalent of 5 mg of copper from Lanthanum chloride : 267 mg/ml of powdered Tablets to a porcelain crucible. Heat for 6 12 lanthanum chloride heptahydrate in N h in a muffle furnace maintained at 550, and cool. Add 15 ml of, and boil gently on a hot Calcium standard stock : 400 µg/ml of calcium plate or a steam bath for 30 min, intermittently rinsing Dissolve g of calcium carbonate, previously dried the inner surface of the crucible with 6 N hydrochloric at 300 for 3 h and cooled in a desiccator for 2 h, and acid. Cool, and quantitatively transfer the contents of the dissolve in 25 ml of 1 N. Boil to expel crucible to a 100-mL volumetric flask, rinsing the carbon dioxide, and dilute with water to 1000 ml. crucible with portions of 6 N. Dilute Standard stock : 100 µg/ml of calcium from the contents of the flask with water to volume, and filter, Calcium standard diluted with N discarding the first 5 ml of the filtrate. Dilute the filtrate quantitatively with N to obtain a Standard s: Into separate 100-mL volumetric concentration of 2 µg/ml of copper. flasks pipet 1.0, 1.5, 2.0, 2.5, and 3.0 ml of the Standard stock. To each flask add 1.0 ml of (See Spectrophotometry and Light-Scattering 851.)

3 USP 35 Dietary Supplements / Calcium 1223 Lamp: Copper hollow-cathode magnesium (Mg) in the portion of Tablets taken: Analytical wavelength: Copper emission line, nm Blank: N C = measured concentration of magnesium in the C U = nominal concentration of magnesium in the Determine the absorbances of the s against the Blank. Plot the absorbances of the Standard s Acceptance criteria: 90.0% 125.0% of the labeled versus concentration, in µg/ml, of copper, and draw amount of magnesium (Mg) the straight line best fitting the five plotted points. MANGANESE, Method 1 From the graph, determine the concentration, C, in µg Manganese standard stock : Transfer 1.00 g of /ml, of copper in the Sample. manganese, weighed, to a 1000-mL volumetric flask. Dissolve in 20 ml of nitric acid, dilute with 6 N Copper (Cu) in the portion of Tablets taken: to volume, and mix to obtain a with a concentration of 1000 µg/ml of manganese. Standard stock : 50 µg/ml of manganese from C = measured concentration of copper in the Manganese standard stock diluted with N C U = nominal concentration of copper in the Standard s: To separate 100-mL volumetric flasks transfer 1.0, 1.5, 2.0, 3.0, and 4.0 ml of the Acceptance criteria: 90.0% 125.0% of the labeled Standard stock. Dilute the contents of each flask amount of copper (Cu) with N to volume to obtain MAGNESIUM, Method 1 s with concentrations of 0.5, 0.75, 1.0, 1.5, and Lanthanum chloride : 267 mg/ml of 2.0 µg/ml of manganese. lanthanum chloride heptahydrate in N Sample : Finely powder NLT 20 Tablets. Transfer the equivalent of 9 mg of manganese from powdered Magnesium standard stock : Transfer 1.00 g of Tablets to a porcelain crucible. Heat for 6 12 h in a magnesium to a 1000-mL volumetric flask, dissolve in 50 muffle furnace maintained at 550, and cool. Add 15 ml ml of 6 N, dilute with water to of, and boil gently on a hot plate or a volume, and mix to obtain a having a known steam bath for 30 min, intermittently rinsing the inner concentration of 1000 µg/ml. surface of the crucible with 6 N. Cool, Standard stock : 20 µg/ml of magnesium from and quantitatively transfer the contents of the crucible to Magnesium standard stock diluted with N a 100-mL volumetric flask, rinsing the crucible with portions of 6 N. Dilute the contents of Standard s: To separate 100-mL volumetric the flask with water to volume, and filter, discarding the flasks transfer 1.0, 1.5, 2.0, 2.5, and 3.0 ml of Standard first 5 ml of the filtrate. Dilute the filtrate quantitatively stock. To each flask add 1.0 ml of Lanthanum with N to obtain a chloride, and dilute with N hydrochloric concentration of 1 µg/ml of manganese. acid to volume to obtain concentrations of 0.2, 0.3, 0.4, 0.5, and 0.6 µg/ml of magnesium. (See Spectrophotometry and Light-Scattering 851.) Sample : Finely powder NLT 20 Tablets. Transfer the equivalent of 200 mg of magnesium to a porcelain Lamp: Manganese hollow-cathode crucible. Heat for 6 12 h in a muffle furnace maintained at 550, and cool. Add 15 ml of, and Analytical wavelength: Manganese emission line, boil gently on a hot plate or a steam bath for 30 min, nm intermittently rinsing the inner surface of the crucible Blank: N with 6 N. Cool, and quantitatively transfer the contents of the crucible to a 100-mL volumetric flask, rinsing the crucible with portions of 6 N Determine the absorbances of the s against the. Dilute the contents of the flask with Blank. Plot the absorbances of the Standard s water to volume, and filter, discarding the first 5 ml of versus the concentration, in µg/ml, of manganese, the filtrate. Dilute the filtrate quantitatively with N and draw the straight line best fitting the five plotted to obtain a concentration of 0.4 µg/ml points. From the graph so obtained, determine the of magnesium, adding 1 ml of Lanthanum chloride concentration, C, in mg/ml, of manganese in the /100 ml of the final volume. Sample. (See Spectrophotometry and Light-Scattering 851.) manganese (Mn) in the portion of Tablets taken: Lamp: Magnesium hollow-cathode Analytical wavelength: Magnesium emission line, C = measured concentration of manganese in the nm Blank: N containing 1 ml of C U = nominal concentration of manganese in the Lanthanum chloride /100 ml Acceptance criteria: 90.0% 125.0% of the labeled amount of manganese Determine the absorbances of the s against the ZINC, Method 1 Blank. Plot the absorbances of the Standard s Zinc standard stock : 1000 µg/ml of zinc from versus concentration, in µg/ml, of magnesium, and zinc oxide dissolved in 5 M (3.89 draw the straight line best fitting the five plotted mg/ml), and diluted with water to final volume. [NOTE points. From the graph, determine the concentration, Dissolve in 5 M by warming, if C, in µg/ml, of magnesium in the Sample. necessary, cool, and then dilute to final volume.]

4 1224 Calcium / Dietary Supplements USP 35 Standard stock : 50 µg/ml of zinc from Zinc Standard s: Prepare a mixture of Standard stock standard stock diluted with N hydrochloric in Diluent to prepare a six-point calibration curve acid to bracket the concentration range of each mineral of Standard s: Transfer 1.0, 2.0, 3.0, 4.0, and 5.0 interest. ml of Standard stock to separate 100-mL Sample : Weigh and finely powder NLT 20 volumentric flasks. Dilute the contents of each flask with Tablets. Transfer a portion equal to the average Tablet N to volume to obtain weight to a 250-mL volumetric flask. Slowly add 25 ml concentrations of 0.5, 1.0, 1.5, 2.0, and 2.5 µg/ml of of Stock aqua regia in 5-mL increments, followed zinc. by mixing. [NOTE If the sample contains a carbonate, Sample : Weigh and finely powder NLT 20 bubbling will occur. Wait until bubbling ends to Tablets. Transfer the equivalent of 40 mg of zinc from proceed.] Bring the to a boil on a hot plate. powdered Tablets to a porcelain crucible. Heat for 6 12 Continue to heat gently until fumes cease (about 1 h). h in a muffle furnace maintained at 550, and cool. Add Remove from heat, cool, and dilute with water to 15 ml of, and boil gently on a hot volume. Filter about 30 ml into a centrifuge tube using plate or a steam bath for 30 min, intermittently rinsing a 5-µm pore size nylon syringe filter. If necessary, make the inner surface of the crucible with 6 N hydrochloric any further dilutions using the Diluent. acid. Cool, and quantitatively transfer the contents of the crucible to a 100-mL volumetric flask, rinsing the (See Spectrochemistry 730.) crucible with portions of 6 N. Dilute Mode: Inductively coupled plasma spectrometry, using the contents of the flask with water to volume, and filter, a spectrometer set to measure the emission of each discarding the first 5 ml of the filtrate. Dilute the filtrate mineral of interest at about the corresponding quantitatively with N to obtain a wavelength. concentration of 2 µg/ml of zinc. [NOTE The operating conditions may be developed and optimized based on the manufacturer s (See Spectrophotometry and Light-Scattering 851.) recommendation. The wavelengths selected should be demonstrated experimentally to provide sufficient Lamp: Zinc hollow-cathode specificity, sensitivity, linearity, accuracy, and precision.] Analytical wavelength: Zinc emission line, nm Blank: N [NOTE Analyze the and obtain the response as directed for.] Determine the absorbances of the s against the Relative standard deviation: NMT 2.0% Blank. Plot the absorbances of the Standard s versus concentration, in µg/ml, of zinc, and draw the straight line best fitting the five plotted points. From Determine the emission of each mineral of interest in the graph, determine the concentration, C, in µg/ml, the Standard s and Sample with an of zinc in the Sample. inductively coupled plasma system using the Diluent as zinc the blank. Plot the emission of the Standard s (Zn) in the portion of Tablets taken: versus concentration, in mg/l, of the minerals of interest, and draw the straight line best fitting the plotted points. From the graph, determine the concentration, C, in mg/l, for each mineral of interest C = measured concentration of zinc in the Sample in the Sample. Calculate the percentage of the (µg/ml) labeled amount for each mineral: C U = nominal concentration of zinc in the Sample (µg/ml) Result = C (V/W) F (W T/L) 100 Acceptance criteria: 90.0% 125.0% of the labeled amount of zinc (Zn) C = measured concentration of the relevant CALCIUM, COPPER, MAGNESIUM, MANGANESE, and ZINC, element in the Sample (mg/l) Method 2 V = volume of the Sample (L) Stock aqua regia : Prepare a mixture of W = sample weight (mg) and nitric acid (3:1) by adding the F = dilution factor of the Sample nitric acid to the. [NOTE Periodically W T = average Tablet weight (mg) vent the in an appropriate fume hood.] L = labeled amount of the relevant element Diluent: Prepare a mixture of Stock aqua regia (mg/tablet) and water (1:9) by adding one volume of Stock aqua Acceptance criteria: NLT 90.0% 125.0% of the labeled regia to two volumes of water. Dilute with amount of calcium (Ca), copper (Cu), magnesium (Mg), additional water to volume, and mix well. manganese (Mn), and zinc (Zn). : Prepare a mixture of 1000 mg/l of yttrium in 5% (v/v) nitric acid and PERFORMANCE TESTS 1000 mg/l of scandium in 5% (v/v) nitric acid DISINTEGRATION AND DISSOLUTION OF DIETARY SUPPLEMENTS with Diluent (1:1:198), and mix : Meet the requirements for Dis with Standard stock (Ca, Cu, Mg, Mn, and Zn): respect to calcium [NOTE It is only necessary to include the minerals of Medium: 0.1 N ; 900 ml interest in the.] Using commercially available Apparatus 2: 75 rpm element standard (single- or multi-element) s in Time: 30 min 5% (v/v) nitric acid, pipet the appropriate : Determine the amount of calcium (Ca) amount of element standard into a volumetric dissolved, using the procedure in the assay for Calcium, flask, and dilute with 5% (v/v) nitric acid to making any necessary volumetric adjustments. obtain a having final concentrations of about Tolerances: NLT 75% of the labeled amount of Ca is 1000 mg/l of calcium, 100 mg/l of copper, 500 mg/l dissolved. of magnesium, 100 mg/l of manganese, and 250 mg/l WEIGHT VARIATION OF DIETARY SUPPLEMENTS 2091 : Meet of zinc. the requirements

5 . fuge Accessed from by newp0rt1 on Wed Nov 30 21:12:01 EST 2011 USP 35 Dietary Supplements / Cat s Claw 1225 SPECIFIC TESTS MICROBIAL ENUMERATION TESTS NUTRITIONAL AND DIETARY SUPPLEMENTS 2021 : The total aerobic microbial count does not exceed 3000 cfu/g, and the total combined molds and yeasts count does not exceed 300 cfu/g. Tablets also meet the requirements of the tests for absence of Salmonella species, Escherichia coli, and Staphylococcus aureus. ABSENCE OF SPECIFIED MICROORGANISMS NUTRITIONAL AND DIETARY SUPPLEMENTS 2022 : Meet the requirements of the tests for absence of Salmonella species, Escherichia coli, and Staphylococcus aureus. Spray reagent B, and examine the plate under daylight. The Sample chromatogram shows multiple orange-brown zones that correspond in color and R F values to those observed in the Standard chromato- gram. Other colored zones of varying intensities may be observed in the Sample. B. The Sample exhibits peaks for speciophylline, uncarine F, mitraphylline, isomitraphylline, pteropodine, and isopteropodine at retention times that correspond to those in Standard A, as obtained in the test for Content of Pentacyclic Oxindole Alkaloids and Limit of Tetra- cyclic Oxindoles. ADDITIONAL REQUIREMENTS COMPOSITION PACKAGING AND STORAGE: Preserve in tight, light-resistant CONTENT OF PENTACYCLIC OXINDOLE ALKALOIDS AND LIMIT containers. OF TETRACYCLIC OXINDOLES LABELING: The label states that the product is Calcium Standard A: Dissolve an accurately weighed and Vitamin D with Minerals Tablets. The label also states quantity of USP Powdered Cat s Claw Extract RS in the quantities of each mineral and vitamin D/dosage methanol, shaking for 1 min. Dilute with methanol to unit, the salt form of the mineral used as the source of obtain a having a known concentration of about each element present, and the chemical form of vitamin 0.5 mg of the labeled amount of total oxindole alkaloids D present in the dosage unit. per ml. Pass through a filter of 0.45-µm or finer pore USP REFERENCE STANDARDS 11 size. USP Cholecalciferol RS Standard B: 0.1 mg/ml of USP Isopteropodine USP Ergocalciferol RS RS in methanol. Pass through a nylon filter of 0.45-µm or finer pore size. Sample : Accurately weigh approximately 750 mg of ground Cat s Claw, and place in a 10-mL centri- Cat s Claw tube. Sonicate with 2.5 ml of methanol for 10 min. Centrifuge, and transfer this to a 10-mL volumetric flask. Repeat the extraction three additional times DEFINITION combining the extracts in the 10-mL volumetric flask, Cat s Claw consists of the inner bark of the stems of Uncaria and dilute with methanol to volume. Transfer about 3 tomentosa (Willd.) DC. (Rubiaceae). It contains NLT 0.3% ml of the to a test tube containing 300 mg of of pentacyclic oxindole alkaloids as isopteropodine, calcunylon filter of 0.45-µm or finer pore size, discarding the polyamide powder, and shake for 1 min. Pass through a lated on the dried basis, as the sum of speciophylline, uncarine F, mitraphylline, isomitraphylline, pteropodine, first part of the filtrate. and isopteropodine. Solution A: Prepare a filtered and degassed 10 mm ph 7.0 phosphate buffer by mixing 6 ml of 1 N sodium IDENTIFICATION hydroxide, 10 ml of 1 M monobasic potassium phos- A. THIN-LAYER CHROMATOGRAPHIC IDENTIFICATION TEST phate, and sufficient water to make 1000 ml. Adjust to Standard : 100 mg of USP Powdered Cat s Claw a ph of 7.0 ± 0.1 by adding more of either. Extract RS in 2 ml of methanol. Sonicate for 5 min, Solution B: Acetonitrile shaking occasionally, heat in a water bath at 60 for 15 Solution C: Methanol and glacial acetic acid (99:1) min, cool, and centrifuge. Mobile phase: See Table 1. Sample : 5 g of powdered Cat s Claw in 10 ml of methanol. Sonicate for 5 min, shaking occasionally. Table 1 Heat the mixture in a water bath at 60 for 15 min, cool, and filter. Time Solution A Solution B Solution C Adsorbent: Chromatographic silica gel mixture with an (min) (%) (%) (%) average particle size of µm (TLC plates) Application volume: 20 µl, as bands that are 1 cm in length Developing solvent system: Ethyl acetate and hexane (95:5) Spray reagent A: Dissolve 0.85 g of basic bismuth ni trate in 10 ml of glacial acetic acid and 40 ml of water by heating. Filter if necessary (Solution A). Dissolve 8 g of potassium iodide in 30 ml of water (Solution B). Mix Solution A and Solution B (1:1) to obtain a stock. Dilute 1 ml of the stock with 2 ml of glacial Chromatographic system acetic acid and 10 ml of water. [NOTE Use freshly mixed Solution A and Solution B.] Spray reagent B: Use a 10% of sodium nitrite Detector: UV 245 nm in water. Column: 4.6-mm 10-cm; end-capped 3-µm packing L1 Samples: Standard and Sample Flow rate: 0.75 ml/min Develop the chromatogram to a length of NLT 12 cm, Injection size: 10 µl and dry the plate in a current of air. Acceptance criteria: Examine the plate under short UV Samples: Standard A and Standard B light. The Sample chromatogram shows multiple zones that correspond in R F values to those observed Chromatogram similarity: The chromatogram obfrom the Standard chromatogram. Other zones tained using Standard A is similar to the Refof varying intensities may be observed in the Sample sodered erence Chromatogram provided with the USP Pow- lution. Spray the plate with Spray reagent A followed by Cat s Claw Extract RS.