Hyoscyami Semen (Unprocessed)(Unprocessed)

HyoscyamiHyoscyami Semen (unprocessed)(unprocessed)

B CC 0.50.5 mm mm

AA 11 mm

FigureFigure 1 1 A A photographphotograph of Hyoscyami Semen (unprocessed)(unprocessed)

A.A. Hyoscyami Hyoscyami SemenSemen (unprocessed)(unprocessed) B. Magnified image of seeds B. Magnified image of seeds C. Magnified image of longitudinal section of seeds C. Magnified image of longitudinal section of seeds

175 175 Hyoscyami Semen (unprocessed) Hyoscyami Semen (unprocessed)

1. NAMES Powder Exotesta cells scattered or in groups, yellow or greyish-yellowish brown, polygonal or elongated Official Name: Hyoscyami Semen (unprocessed) polygonal in surface view, 75-311 µm long, 25-123 µm in diameter, anticlinal walls thickened,

Chinese Name: undulant, with distinct striations, containing yellowish-brown contents; bright yellowish-white under the polarized microscope. Endosperm cells polygonal or subrounded in surface view, cell Chinese Phonetic Name: Tianxianzi (Sheng) walls slightly thickened, containing aleurone grains and oil droplets. Cotyledon cells colourless, subpolygonal, with thin walls, containing oil droplets (Fig. 3).

2. SOURCE

Hyoscyami Semen (unprocessed) is the unprocessed dried ripe seed of Hyoscyamus niger L. (Solanaceae). The fruit is collected in summer and autumn when the pericarp turns yellow, exposed to the sun, afterwards the seeds tapped out, pericarp and stalk removed, then dried under the sun to obtain Hyoscyami Semen (unprocessed).

3. DESCRIPTION

Flattened-subreniform to flattened-ovoid, about 1 mm in diameter. Externally brownish-yellow or greyish-yellow, with fine and dense reticulate striations, and a pitted hilum located at the slightly acute end. Cut surface greyish-white, oily, containing endosperm, embryo curved. Odour slight (Fig. 1).

4. IDENTIFICATION

4.1 Microscopic Identification (Appendix III)

Transverse section Exotesta cells with irregularly undulant protuberances, apex of the protuberances tapering or blunt, cell walls with transparent striations. Endotesta consists of 1 layer of cells, cells mostly tangentially elongated, with thin and slightly wizened walls, containing brown contents. Endosperm cells with slightly thickened walls, containing aleurone grains. Cotyledon cells with thin walls, containing oil droplets (Fig. 2).

176 177 Hyoscyami Semen (unprocessed) Hyoscyami Semen (unprocessed)

2. SOURCE

3. DESCRIPTION

4. IDENTIFICATION

4.1 Microscopic Identification (Appendix III)

176 177 Hyoscyami Semen (unprocessed) Hyoscyami Semen (unprocessed)

1a 1b 1 2 1 3

4 2 3

2a 3a 1 2 3 4 4

B 100 μm C 25 μm 50 μm

Figure 2 Microscopic features of transverse section of Hyoscyami Semen (unprocessed) Figure 3 Microscopic features of powder of Hyoscyami Semen (unprocessed)

A. Sketch B. Section illustration C. Section magnified 1. Exotesta cells 2. Endosperm cells (oil droplets , aleurone grains ) 3. Cotyledon cells (oil droplets ) 1. Exotesta 2. Endotesta 3. Endosperm 4. Cotyledon a. Features under the light microscope b. Features under the polarized microscope

178 179 Hyoscyami Semen (unprocessed) Hyoscyami Semen (unprocessed)

1a 1b 1 2 1 3

4 2 3

2a 3a 1 2 3 4 4

B 100 μm C 25 μm 50 μm

Figure 2 Microscopic features of transverse section of Hyoscyami Semen (unprocessed) Figure 3 Microscopic features of powder of Hyoscyami Semen (unprocessed)

178 179 Hyoscyami Semen (unprocessed) Hyoscyami Semen (unprocessed)

4.2 Thin-Layer Chromatographic Identification [Appendix IV(A)] Procedure Carry out the method by using a HPTLC silica gel G60 plate, a twin trough chamber and a Standard solutions freshly prepared developing solvent system as described above. Apply separately L-hyoscyamine L-Hyoscyamine sulphate dihydrate standard solution sulphate dihydrate standard solution (3 μL), scopolamine hydrobromide standard solution Weigh 1.0 mg of L-hyoscyamine sulphate dihydrate CRS (Fig. 4) and dissolve in 1 mL of (3 μL) and the test solution (5 μL) to the plate. Before the development, add the developing methanol. Keep at about 4ºC. solvent to one of the troughs of the chamber and place the HPTLC plate in the other trough. Scopolamine hydrobromide standard solution Cover the chamber with a lid and let equilibrate for about 15 min. Carefully tilt the chamber to Weigh 1.0 mg of scopolamine hydrobromide CRS (Fig. 4) and dissolve in 1 mL of methanol. allow sufficient solvent to pass from the trough containing the solvent to the other containing Keep at about 4ºC. the HPTLC plate for development. Develop over a path of about 8 cm. After the development,

Developing solvent system remove the plate from the chamber, mark the solvent front and heat at about 105ºC (about 10 min). Prepare a mixture of ammonium hydroxide solution (25%, v/v), methanol and ethyl acetate Spray the plate evenly with the spray reagent 1 and the spray reagent 2, then dry in air. Examine (0.5:1:8.5, v/v). the plate under visible light. Calculate the Rf values by using the equation as indicated in Appendix IV (A). Spray reagent Solution A (i) Weigh 0.85 g of bismuth oxynitrate and dissolve in a mixture of 10 mL of glacial acetic acid and 40 mL of water. Solution B Weigh 4 g of potassium iodide and dissolve in 10 mL of water. Spray reagent 1 Transfer 5 mL of Solution A, 5 mL of Solution B and 20 mL of glacial acetic acid into a 100-mL volumetric flask and make up to the mark with water. Freshly prepare the reagent. Spray reagent 2 Weigh 5 g of sodium nitrite and dissolve in 50 mL of ethanol (60%).

Test solution Weigh 2.0 g of the powdered sample and place it in a 100-mL conical flask, then add 15 mL (ii) of petroleum ether (30-60ºC). Sonicate (160 W) the mixture for 20 min. Filter and discard the filtrate. Repeat the extraction for one more time. Dissolve the residue in 2 mL ammonium hydroxide solution (25%, v/v) and 30 mL of ethanol. Sonicate (160 W) the mixture for 30 min. Filter and transfer the filtrate to a 100-mL round-bottomed flask. Wash the residue for three times each with 10 mL of ethanol. Evaporate the solvent to dryness at reduced pressure in a rotary evaporator. Dissolve the residue in 1 mL of methanol. Figure 4 Chemical structures of (i) L-hyoscyamine sulphate dihydrate and (ii) scopolamine hydrobromide

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Front Standard solutions L-Hyoscyamine sulphate dihydrate standard solution for fingerprinting, Std-FP (20 mg/L) Weigh 0.2 mg of L-hyoscyamine sulphate dihydrate CRS and dissolve in 10 mL of methanol. Keep at about 4ºC. Scopolamine hydrobromide standard solution for fingerprinting, Std-FP (20 mg/L) Weigh 0.2 mg of scopolamine hydrobromide CRS and dissolve in 10 mL of methanol. Keep at about 4ºC.

Test solution Weigh 1.0 g of the powdered sample and place it in a cellulose extraction thimble. Place the cellulose extraction thimble in soxhlet extractor. Add 200 mL of petroleum ether (30-60ºC) to a 250-mL round-bottomed flask. Perform the soxhlet extraction for 2 h. Discard Start the petroleum ether and dry the residue in air. Dissolve the residue in 5 mL of ammonium 1 2 3 hydroxide solution (25%, v/v) and 75 mL of ethanol. Reflux the mixture for 2 h. Cool down to room temperature. Filter and transfer the filtrate to a 100-mL volumetric flask. Wash the Figure 5 A reference HPTLC chromatogram of Hyoscyami Semen (unprocessed) extract observed under residue with ethanol. Combine the solution and make up to the mark with ethanol. Pipette visible light after staining 20 mL of the solution to a 50-mL conical flask. Adjust the pH to 5 with hydrochloric acid solution (10%, v/v). Pre-condition a solid-phase extraction column (strong cation exchange, 1. Scopolamine hydrobromide standard solution 6 mL, 500 mg) with 3 mL of methanol, 3 mL of a mixture of ammonium hydroxide solution 2. L-Hyoscyamine sulphate dihydrate standard solution (25%, v/v) and ethanol (1:15, v/v), then followed by 3 mL of ethanol and 5 mL of ethanol 3. Test solution [adjust the pH to 4 with hydrochloric acid solution (10%, v/v)]. Allow to stand for 10 min. Load the sample solution to the pre-conditioned SPE column. Add 3 mL of ethanol to the For positive identification, the sample must give spots or bands with chromatographic characteristics, column and discard the eluant. Add 5 mL of a mixture of ammonium hydroxide solution including the colour and the Rf values, corresponding to those of L-hyoscyamine and scopolamine (25%, v/v) and ethanol (1:15, v/v) to the column. Collect the eluant in a 100-mL round-bottomed (Fig. 5). flask. Evaporate the solvent to near dryness at reduced pressure in a rotary evaporator. Dissolve the residue in methanol. Transfer the solution to a 2-mL volumetric flask and make up to the 4.3 High-Performance Liquid Chromatographic Fingerprinting (Appendix XII) mark with methanol. Filter through a 0.45-µm nylon filter.

Reagents Chromatographic system 0.005 M Sodium 1-heptanesulphonate solution The liquid chromatograph is equipped with a DAD (210 nm) and a column (4.6 × 250 mm) Weigh 0.5 g of sodium 1-heptanesulphonate and dissolve in 500 mL of water. packed with ODS bonded silica gel (5 µm particle size). The column temperature is maintained at 0.01 M Potassium dihydrogen phosphate solution 35ºC during the separation. The flow rate is about 1.0 mL/min. Programme the chromatographic Weigh 0.68 g of potassium dihydrogen phosphate and dissolve in 500 mL of water. system as follows (Table 1) – Sodium 1-heptanesulphonate - potassium dihydrogen phosphate buffer solution (pH 5) Transfer 500 mL of 0.005 M sodium 1-heptanesulphonate solution and 500 mL of 0.01 M potassium dihydrogen phosphate solution to a 1500-mL conical flask. Adjust the pH to 5 with 1.6 M potassium hydroxide solution.

182 183 Hyoscyami Semen (unprocessed) Hyoscyami Semen (unprocessed)

Test solution Weigh 1.01.0 gg ofof thethe powderedpowdered sample sample and and place place it itin ina cellulosea cellulose extraction extraction thimble. thimble. Place Place the cellulosethe cellulose extraction extraction thimble thimble in soxhlet in extractor.soxhlet extractor. Add 200 mLAdd of 200 petroleum mL of ether petroleum (30-60ºC) ether to a(30-60ºC) 250-mL round-bottomedto a 250-mL round-bottomed flask. Perform flask. the soxhletPerform extraction the soxhlet for extraction 2 h. Discard for the2 h. petroleum Discard Start etherthe petroleum (30-60ºC) etherand dry and the dry residue the residue in air. Transfer in air. Dissolve the dried theresidue residue to a 250-mLin 5 mL round-bottomed of ammonium 1 2 3 flaskhydroxide and addsolution 5 mL (25%, of ammonium v/v) and 75hydroxide mL of ethanol.solution (25%,Reflux v/v)the andmixture 75 mLfor of2 ethanol.h. Cool downReflux theto room mixture temperature. for 2 h. Cool Filter down and to transfer room temperature. the filtrate Filterto a 100-mL and transfer volumetric the filtrate flask. to Washa 100-mL the Figure 5 A reference HPTLC chromatogram of Hyoscyami Semen (unprocessed) extract observed under volumetricresidue with flask. ethanol. Wash Combine the residue the withsolution ethanol. and Combinemake up the to solutionthe mark and with make ethanol. up to the Pipette mark visible light after staining with20 mL ethanol. of the Pipettesolution 20 to mL a 50-mLof the solutionconical flask.to a 50-mL Adjust conical the pH flask. to 5 withAdjust hydrochloric the pH to 5 acidwith hydrochloricsolution (10%, acid v/v). solution Pre-condition (10%, v/v). a solid-phase Pre-condition extraction a solid-phase column extraction (strong cation column exchange, (strong 1. Scopolamine hydrobromide standard solution cation6 mL, 500exchange, mg) with 6 mL, 3 mL 500 of mg)methanol, with 3 3 mL mL of of methanol, a mixture 3of mL ammonium of a mixture hydroxide of ammonium solution 2. L-Hyoscyamine sulphate dihydrate standard solution hydroxide(25%, v/v) solution and ethanol (25%, (1:15, v/v) and v/v), ethanol then followed(1:15, v/v), by then 3 mL followed of ethanol by 3 and mL 5of mL ethanol of ethanol and 5 3. Test solution mL[adjust of ethanolthe pH [adjustto 4 with the hydrochloricpH to 4 with acidhydrochloric solution acid(10%, solution v/v)]. Allow(10%, v/v)].to stand Allow for to10 standmin. forLoad 10 themin. sample Load thesolution sample to solution the pre-conditioned to the pre-conditioned SPE column. SPE column.Add 3 mL Add of 3 ethanolmL of ethanol to the For positive identification, the sample must give spots or bands with chromatographic characteristics, tocolumn the column and discard and discard the eluant. the eluant. Add Add 5 mL 5 mLof aof mixture a mixture of ofammonium ammonium hydroxide hydroxide solutionsolution including the colour and the Rf values, corresponding to those of L-hyoscyamine and scopolamine (25%, v/v) and ethanol (1:15, v/v) to the column. Collect the eluant in a 100-mL round-bottomed (Fig. 5). flask. EvaporateEvaporate thethe solventsolvent toto nearnear drynessdryness atat reducedreduced pressurepressure inin aa rotaryrotary evaporator.evaporator. DissolveDissolve the residue in methanol. Transfer the solution to a 2-mL volumetric flask and make up to the 4.3 High-Performance Liquid Chromatographic Fingerprinting (Appendix XII) mark with methanol. Filter through a 0.45-μm0.45-µm nylon filter.

Reagents Chromatographic system 0.005 M Sodium 1-heptanesulphonate solution The liquid chromatograph isis equippedequipped withwith aa DADDAD (210(210 nm)nm) andand aa columncolumn (4.6(4.6 ×× 250 mm) Weigh 0.5 g of sodium 1-heptanesulphonate and dissolve in 500 mL of water. packed with ODS bonded silica gel (5 µmμm particle size). The column temperature is maintained at 0.01 M Potassium dihydrogen phosphate solution 35ºC during the separation. The flow raterate isis aboutabout 1.01.0 mL/min.mL/min. ProgrammeProgramme thethe chromatographicchromatographic Weigh 0.68 g of potassium dihydrogen phosphate and dissolve in 500 mL of water. system asas followsfollows (Table (Table 1) 1) – – Sodium 1-heptanesulphonate - potassium dihydrogen phosphate buffer solution (pH 5) Transfer 500 mL of 0.005 M sodium 1-heptanesulphonate solution and 500 mL of 0.01 M potassium dihydrogen phosphate solution to a 1500-mL conical flask. Adjust the pH to 5 with 1.6 M potassium hydroxide solution.

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Table 1 Chromatographic system conditions The RRTs and acceptable ranges of the four characteristic peaks of Hyoscyami Semen (unprocessed) extract are listed in Table 2. Sodium Time 1-heptanesulphonate - potassium Acetonitrile (min) dihydrogen phosphate buffer Elution Table 2 The RRTs and acceptable ranges of the four characteristic peaks of Hyoscyami Semen (%, v/v) solution (pH 5) (unprocessed) extract (%, v/v) Peak No. RRT Acceptable Range 0 – 10 90 → 82 10 → 18 linear gradient 1 0.54 ± 0.03 10 – 30 82 → 77 18 → 23 linear gradient 2 0.61 ± 0.03 30 – 35 77 23 isocratic 3 (marker, scopolamine) 1.00 - 35 – 40 77 → 73 23 → 27 linear gradient 4 (L-hyoscyamine) 1.27 ± 0.03

System suitability requirements Perform at least five replicate injections, each using 5 µL of L-hyoscyamine sulphate dihydrate Std-FP and scopolamine hydrobromide Std-FP. The requirements of the system suitability parameters are as follows: the RSD of the peak areas of L-hyoscyamine and scopolamine should not be more than 5.0%; the RSD of the retention times of L-hyoscyamine and scopolamine peaks should not be more than 2.0%; the column efficiencies determined from L-hyoscyamine and scopolamine peaks should not be less than 20000 theoretical plates.

The R value between peak 3 and the closest peak; and the R value between peak 4 and the closest peak in the chromatogram of the test solution should not be less than 1.5 (Fig. 6).

Procedure Separately inject L-hyoscyamine sulphate dihydrate Std-FP, scopolamine hydrobromide Std-FP and the test solution (5 µL each) into the HPLC system and record the chromatograms. Measure the retention times of L-hyoscyamine and scopolamine peaks in the chromatograms of L-hyoscyamine sulphate dihydrate Std-FP, scopolamine hydrobromide Std-FP and the retention times of the four Figure 6 A reference fingerprint chromatogram of Hyoscyami Semen (unprocessed) extract characteristic peaks (Fig. 6) in the chromatogram of the test solution. Identify L-hyoscyamine For positive identification, the sample must give the above four characteristic peaks with RRTs and scopolamine peaks in the chromatogram of the test solution by comparing its retention time falling within the acceptable range of the corresponding peaks in the reference fingerprint with that in the chromatograms of L-hyoscyamine sulphate dihydrate Std-FP and scopolamine chromatogram (Fig. 6). hydrobromide Std-FP. The retention times of L-hyoscyamine and scopolamine peaks in the chromatograms of the test solution and the corresponding Std-FP should not differ by more than 2.0%. Calculate the RRTs of the characteristic peaks by using the equation as indicated in Appendix XII.

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The R value between peak 3 and the closest peak; and the R value between peak 4 and the closest peak in the chromatogram of the test solution should not be less than 1.5 (Fig. 6).

184 185 Hyoscyami Semen (unprocessed) Hyoscyami Semen (unprocessed)

Hyoscyami Semen (unprocessed) 5. TESTS Standard solution Mixed L-hyoscyamine sulphate dihydrate and scopolamine hydrobromide standard stock solution, (Appendix V): meet the requirements. 5. TESTS5.1 Heavy Metals Std-Stock (200 mg/L each) Weigh accurately 2.0 mg of L-hyoscyamine sulphate dihydrate CRS and scopolamine hydrobromide 5.15.2 HeavyPesticid Me etalsResidues (Appendix (Appendix V): meet VI): themeet requirements. the requirements. CRS each, and dissolve in 10 mL of methanol. Keep at about 4ºC. 5.25.3 PesticidMycotoxinse Residues (Appendix (Appendix VII): meet VI) the: meet requirements. the requirements. Mixed L-hyoscyamine sulphate dihydrate and scopolamine hydrobromide standard solution for assay, Std-AS 5.45.3 MycotoxinsForSulphureign MatterDioxide (Appendix (Appendix Residues VII): VIII):meet (Appendix the not requirements. more XVI) than: meet 3.0%. the requirements. Measure accurately the volume of the mixed L-hyoscyamine sulphate dihydrate and scopolamine

5.55.4 ForAsheign (Appendix Matter IX) (Appendix VIII): not more than 3.0%. hydrobromide Std-Stock, dilute with methanol to produce a series of solutions of 10, 60, 120, 160, 200 mg/L for both L-hyoscyamine sulphate dihydrate and scopolamine hydrobromide. Keep at about 4ºC. 5.65.5 AshTotal (Appendixash: not more IX) than 6.5%. Acid-insoluble ash: not more than 2.5%. Test solution Total ash: not more than 6.5%. Weigh accurately 1.0 g of the powdered sample and place it in a cellulose extraction thimble. Place 5.6 WAcid-insolubleater Content ash: (Appendix not more X)than 2.5%. the cellulose extraction thimble in soxhlet extractor. Add 200 mL of petroleum ether (30-60ºC) to a 250-mL round-bottomed flask. Perform the soxhlet extraction for 2 h. Discard the petroleum ether and 5.75.6 WOvenater dried Content method: (Appendix not more X)than 8.0%. dry the residue in air. Dissolve the residue in 5 mL of ammonium hydroxide solution (25%, v/v) and Oven dried method: not more than 8.0%. 75 mL of ethanol. Reflux the mixture for 2 h. Cool down to room temperature. Filter and transfer the filtrate to a 100-mL volumetric flask. Wash the residue with ethanol. Combine the solution and make 6. EXTRACTIVES (Appendix XI) up to the mark with ethanol. Pipette 20 mL of the solution to a 50-mL conical flask. Adjust the pH 6. EXTRACTIVESWater-soluble extractives (Appendix (cold extractionXI) method): not less than 8.0%. to 5 with hydrochloric acid solution (10%, v/v). Pre-condition a solid-phase extraction column Ethanol-soluble extractives (cold extraction method): not less than 14.0%. (strong cation exchange, 6 mL, 500 mg) with 3 mL of methanol, 3 mL of a mixture of ammonium Water-soluble extractives (cold extraction method): not less than 8.0%. hydroxide solution (25%, v/v) and ethanol (1:15, v/v), then followed by 3 mL of ethanol and 5 mL of Ethanol-soluble extractives (cold extraction method): not less than 14.0%. ethanol [adjust the pH to 4 with hydrochloric acid solution (10%, v/v)]. Allow to stand for 10 min. 7. ASSAY Load the sample solution to the pre-conditioned SPE column. Add 3 mL of ethanol to the column and discard the eluant. Add 5 mL of a mixture of ammonium hydroxide solution (25%, v/v) and 7. ASSAY Carry out the method as directed in Appendix IV (B). ethanol (1:15, v/v) to the column. Collect the eluant in a 100-mL round-bottomed flask. Evaporate the solvent to near dryness at reduced pressure in a rotary evaporator. Dissolve the residue in methanol. Carry out the method as directed in Appendix IV (B). Reagents Transfer the solution to a 2-mL volumetric flask and make up to the mark with methanol. Filter 0.005 M Sodium 1-heptanesulphonate solution through a 0.45-µm nylon filter. WReagentseigh 0.5 g of sodium 1-heptanesulphonate and dissolve in 500 mL of water. 0.010.005 M M Potassium Sodium 1-heptanesulphonate dihydrogen phosphate solution solution Weigh 0.680.5 g g of of sodium potassium 1-heptanesulphonate dihydrogen phosphate and dissolve and dissolve in 500 in mL 500 of mL water. of water. Sodium0.01 M Potassium1-heptanesulphonate dihydrogen - phosphatepotassium solutiondihydrogen phosphate buffer solution (pH 5) TransferWeigh 0.68 500 g mLof potassium of 0.005 Mdihydrogen sodium 1-heptanesulphonate phosphate and dissolve solution in 500 and mL 500 of water.mL of 0.01 M potassium dihydrogenSodium 1-heptanesulphonate phosphate solution - potassiumto a 1500-mL dihydrogen conical phosphateflask. Adjust buffer the solutionpH to 5 (pHwith 5) 1.6 M potassium hydroxideTransfer 500 solution. mL of 0.005 M sodium 1-heptanesulphonate solution and 500 mL of 0.01 M potassium dihydrogen phosphate solution to a 1500-mL conical flask. Adjust the pH to 5 with 1.6 M potassium hydroxide solution.

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Hyoscyami Semen (unprocessed) 5. TESTS Standard solution Mixed L-hyoscyamine sulphate dihydrate and scopolamine hydrobromide standard stock solution, (Appendix V): meet the requirements. 5. TESTS5.1 Heavy Metals Std-Stock (200 mg/L each) Weigh accurately 2.02.0 mg mg of of L L-hyoscyamine-hyoscyamine sulphate sulphate dihydrate dihydrate CRS CRS and and scopolamine 2.0 mg of hydrobromide scopolamine 5.15.2 HeavyPesticid Me etalsResidues (Appendix (Appendix V): meet VI): themeet requirements. the requirements. hydrobromideCRS each, and CRS, dissolve and in dissolve 10 mL inof 10methanol. mL of methanol. Keep at about Keep 4ºC. at about 4ºC. 5.25.3 PesticidMycotoxinse Residues (Appendix (Appendix VII): meet VI) the: meet requirements. the requirements. Mixed L-hyoscyamine sulphate dihydrate and scopolamine hydrobromide standard solution for assay, Std-AS 5.35.4 MycotoxinsForeign Matter (Appendix (Appendix VII): VIII):meet the not requirements. more than 3.0%. Measure accurately the volume of the mixed L-hyoscyamine sulphate dihydrate and scopolamine

5.45.5 ForAsheign (Appendix Matter IX) (Appendix VIII): not more than 3.0%. hydrobromide Std-Stock, dilute with methanol to produce a series of solutions of 10, 60, 120, 160, 200 mg/L for both L-hyoscyamine sulphate dihydrate and scopolamine hydrobromide. Keep at about 4ºC. 5.5 AshTotal (Appendixash: not more IX) than 6.5%. Acid-insoluble ash: not more than 2.5%. Test solution Total ash: not more than 6.5%. Weigh accurately 1.0 g of the powdered sample and place it in aa cellulosecellulose extractionextraction thimble. Place 5.6 WAcid-insolubleater Content ash: (Appendix not more X)than 2.5%. the cellulose extraction thimble in soxhlet extractor. AddAdd 200 mL of petroleum ether (30-60ºC) to a 250-mL round-bottomedround-bottomed flask. flask. Perform Perform the the soxhlet soxhlet extraction extraction for for2 h. 2Discard h. Discard the petroleumthe petroleum ether etherand 5.6 WOvenater dried Content method: (Appendix not more X)than 8.0%. (30-60ºC)dry the residue and dryin air. the Dissolve residue thein air. residue Transfer in 5 themL driedof ammonium residue to hydroxide a 250-mL solution round-bottomed (25%, v/v) flask and Oven dried method: not more than 8.0%. and75 mL add of 5 ethanol.mL of ammonium Reflux the hydroxidemixture for solution 2 h. Cool (25%, down v/v) to and room 75 temperature.mL of ethanol. Filter Reflux and transferthe mixture the forfiltrate 2 h. to Cool a 100-mL down tovolumetric room temperature. flask. Wash Filter the residueand transfer with theethanol. filtrate Combine to a 100-mL the solution volumetric and make flask. 6. EXTRACTIVES (Appendix XI) Washup to the markresidue with with ethanol. ethanol. Pipette Combine 20 mLthe ofsolution the solution and make to a up50-mL to the conical mark withflask. ethanol. Adjust Pipettethe pH 6. EXTRACTIVESWater-soluble extractives (Appendix (cold extractionXI) method): not less than 8.0%. 20to 5mL with of thehydrochloric solution to acida 50-mL solution conical (10%, flask. v/v). Adjust Pre-condition the pH to 5a solid-phasewith hydrochloric extraction acid columnsolution Ethanol-soluble extractives (cold extraction method): not less than 14.0%. (10%,(strong v/v). cation Pre-condition exchange, 6 a mL, solid-phase 500 mg) extraction with 3 mL column of methanol, (strong 3cation mL of exchange, a mixture 6 ofmL, ammonium 500 mg) Water-soluble extractives (cold extraction method): not less than 8.0%. withhydroxide 3 mL solution of methanol, (25%, v/v)3 mL and of ethanol a mixture (1:15, of v/v), ammonium then followed hydroxide by 3 mL solution of ethanol (25%, and v/v) 5 mL and of Ethanol-soluble extractives (cold extraction method): not less than 14.0%. ethanol [adjust(1:15, v/v), the pHthen to followed4 with hydrochloric by 3 mL of acidethanol solution and 5 (10%, mL of v/v)]. ethanol Allow [adjust to standthe pH for to 10 4 min.with 7. ASSAY hydrochloricLoad the sample acid solutionsolution to(10%, the pre-conditionedv/v)]. Allow to standSPE column.for 10 min. Add Load 3 mL the of sampleethanol solutionto the column to the pre-conditionedand discard the SPEeluant. column. Add 5Add mL 3of mL a mixtureof ethanol of toammonium the column hydroxide and discard solution the eluant. (25%, Add v/v) 5 andmL 7. ASSAY Carry out the method as directed in Appendix IV (B). ofethanol a mixture (1:15, of v/v) ammonium to the column. hydroxide Collect solution the eluant (25%, in a v/v)100-mL and round-bottomed ethanol (1:15, v/v)flask. to Evaporate the column. the Collectsolvent theto near eluant dryness in a 100-mL at reduced round-bottomed pressure in aflask. rotary Evaporate evaporator. the Dissolve solvent to the near residue dryness in methanol.at reduced Carry out the method as directed in Appendix IV (B). Reagents pressureTransfer inthe a solutionrotary evaporator. to a 2-mL Dissolvevolumetric the flask residue and in make methanol. up to Transferthe mark the with solution methanol. to a Filter2-mL 0.005 M Sodium 1-heptanesulphonate solution volumetricthrough a 0.45-µm flask and nylon make filter. up to the mark with methanol. Filter through a 0.45-μm nylon filter. WReagentseigh 0.5 g of sodium 1-heptanesulphonate and dissolve in 500 mL of water. 0.010.005 M M Potassium Sodium 1-heptanesulphonate dihydrogen phosphate solution solution Weigh 0.680.5 g g of of sodium potassium 1-heptanesulphonate dihydrogen phosphate and dissolve and dissolve in 500 in mL 500 of mL water. of water. Sodium0.01 M Potassium1-heptanesulphonate dihydrogen - phosphatepotassium solutiondihydrogen phosphate buffer solution (pH 5) TransferWeigh 0.68 500 g mLof potassium of 0.005 Mdihydrogen sodium 1-heptanesulphonate phosphate and dissolve solution in 500 and mL 500 of water.mL of 0.01 M potassium dihydrogenSodium 1-heptanesulphonate phosphate solution - potassiumto a 1500-mL dihydrogen conical phosphateflask. Adjust buffer the solutionpH to 5 (pHwith 5) 1.6 M potassium hydroxideTransfer 500 solution. mL of 0.005 M sodium 1-heptanesulphonate solution and 500 mL of 0.01 M potassium dihydrogen phosphate solution to a 1500-mL conical flask. Adjust the pH to 5 with 1.6 M potassium hydroxide solution.

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Chromatographic system Procedure The liquid chromatograph is equipped with a DAD (210 nm) and a column (4.6 × 250 mm) packed Inject 5 µL of the test solution into the HPLC system and record the chromatogram. Identify with ODS bonded silica gel (5 µm particle size). The column temperature is maintained at 35ºC during L-hyoscyamine and scopolamine peaks in the chromatogram of the test solution by comparing their the separation. The flow rate is about 1.0 mL/min. Programme the chromatographic system as follows retention times with those in the chromatogram of the mixed L-hyoscyamine sulphate dihydrate and (Table 3) – scopolamine hydrobromide Std-AS. The retention times of L-hyoscyamine and scopolamine peaks in the chromatograms of the test solution and the Std-AS should not differ by more than 5.0%. Measure Table 3 Chromatographic system conditions the peak areas and calculate the concentrations (in milligram per litre) of L-hyoscyamine sulphate dihydrate and scopolamine hydrobromide in the test solution, and calculate the percentage contents of Sodium Time 1-heptanesulphonate - potassium L-hyoscyamine (the percentage content of L-hyoscyamine sulphate dihydrate × 0.41, where 0.41 is the Acetonitrile (min) dihydrogen phosphate buffer solution Elution (%, v/v) molar mass ratio of L-hyoscyamine and L-hyoscyamine sulphate dihydrate) and scopolamine (pH 5) (%, v/v) (the percentage content of scopolamine hydrobromide × 0.79, where 0.79 is the molar mass ratio of scopolamine and scopolamine hydrobromide) in the sample by using the equations as indicated in 0 – 10 90 → 82 10 → 18 linear gradient Appendix IV (B). 10 – 30 82 → 77 18 → 23 linear gradient 30 – 35 77 23 isocratic Limits

35 – 40 77 → 73 23 → 27 linear gradient The sample contains not less than 0.018% of L-hyoscyamine (C17H23NO3) and not less than 0.026% of

scopolamine (C17H21NO4), calculated with reference to the dried substance. System suitability requirements Perform at least five replicate injections, each using 5 µL of the mixed L-hyoscyamine sulphate dihydrate and scopolamine hydrobromide Std-AS (120 mg/L each). The requirements of the system 8. CAUTIONS suitability parameters are as follows: the RSD of the peak areas of L-hyoscyamine and scopolamine (1) This CMM is potent / toxic. should not be more than 5.0%; the RSD of the retention times of L-hyoscyamine and scopolamine (2) Should be prescribed by registered Chinese medicine practitioner. peaks should not be more than 2.0%; the column efficiencies determined from L-hyoscyamine and scopolamine peaks should not be less than 20000 theoretical plates.

The R value between L-hyoscyamine peak and the closest peak; and the R value between scopolamine peak and the closest peak in the chromatogram of the test solution should not be less than 1.5.

Calibration curves Inject a series of the mixed L-hyoscyamine sulphate dihydrate and scopolamine hydrobromide Std-AS (5 µL each) into the HPLC system and record the chromatograms. Plot the peak areas of L-hyoscyamine and scopolamine against the corresponding concentrations of the mixed L-hyoscyamine sulphate dihydrate and scopolamine hydrobromide Std-AS. Obtain the slopes, y-intercepts and the r2 values from the corresponding 5-point calibration curves.

188 189 Hyoscyami Semen (unprocessed) Hyoscyami Semen (unprocessed)

35 – 40 77 → 73 23 → 27 linear gradient The sample contains not less than 0.018% of L-hyoscyamine (C17H23NO3) and not less than 0.026% of

scopolamine (C17H21NO4), calculated with reference to the dried substance. System suitability requirements Perform at least five replicate injections, each using 5 µL of the mixed L-hyoscyamine sulphate dihydrate and scopolamine hydrobromide Std-AS (120 mg/L each). The requirements of the system 8. CAUTIONS suitability parameters are as follows: the RSD of the peak areas of L-hyoscyamine and scopolamine (1)This ThisCMM CMM is potent/toxic is potent / andtoxic. should be prescribed by registered Chinese medicine practitioner. should not be more than 5.0%; the RSD of the retention times of L-hyoscyamine and scopolamine (2) Should be prescribed by registered Chinese medicine practitioner. peaks should not be more than 2.0%; the column efficiencies determined from L-hyoscyamine and scopolamine peaks should not be less than 20000 theoretical plates.

The R value between L-hyoscyamine peak and the closest peak; and the R value between scopolamine peak and the closest peak in the chromatogram of the test solution should not be less than 1.5.

188 189