检测：按上述色谱条件，分别精密量取0.5µL供试品溶液和对照品溶液进样

检测：按上述色谱条件，分别精密量取0.5µL供试品溶液和对照品溶液进样，进行含量测定。
2.1.2 实验结果



供试品溶液色谱图

对照品溶液色谱图
色谱图中各成分的分离度及拖尾因子如下：

脂肪酸甲酯 对照品溶液 供试品溶液
分离度 拖尾因子 分离度 拖尾因子
正己酸甲酯 32.00 0.97 24.31 0.96
辛酸甲酯 40.01 1.00 36.30 1.00
癸酸甲酯 45.90 1.04 45.02 1.01
月桂酸甲酯 40.59 1.51 40.78 1.43
肉豆蔻酸甲酯 32.88 1.17 33.55 1.13
棕榈酸甲酯 30.71 1.21 31.29 1.15
棕榈油酸甲酯 4.54 1.13 4.54 1.07
十七酸甲酯 10.82 1.09 11.16 1.09
硬脂酸甲酯 14.36 1.04 14.66 1.03
油酸甲酯 3.55 1.65 3.59 1.68
亚油酸甲酯 6.61 1.16 6.60 1.14
亚麻酸甲酯 9.71 1.02 9.75 1.03
结果表明：对照品图谱与供试品图谱各个组分均符合系统适用性要求，同时从供试品图谱可知，月桂酸、肉豆蒄酸、棕榈酸、油酸及亚油酸含量较高，其它脂肪酸含量较低。
同时进行了对照品溶液的进样精密度试验，结果各组分连续进样6次，组分与内标的响应之比的RSD值最大为1.38 %，供试品溶液进行了室温稳定性考察，结果室温放置8h，各组分与内标的响应之比RSD值最大为2.29 %（亚麻酸甲酯），基本符合测定要求，数据如下表：



精密度考察结果
脂肪酸甲酯 1# 2# 3# 4# 5# 6# 平均 RSD(%)
正己酸甲酯 1.14 1.14 1.14 1.13 1.14 1.14 1.14 0.26
辛酸甲酯 1.30 1.30 1.30 1.29 1.30 1.30 1.30 0.26
癸酸甲酯 13.01 12.99 12.97 12.92 13.01 12.97 12.98 0.25
月桂酸甲酯 5.22 5.22 5.21 5.20 5.22 5.21 5.21 0.17
肉豆蔻酸甲酯 4.87 4.87 4.86 4.85 4.87 4.86 4.86 0.15
棕榈酸甲酯 0.09 0.09 0.09 0.09 0.09 0.09 0.09 1.38
棕榈油酸甲酯 2.76 2.76 2.75 2.75 2.76 2.76 2.76 0.14
硬脂酸甲酯 17.97 17.98 17.93 17.93 17.98 17.97 17.96 0.13
油酸甲酯 5.41 5.41 5.39 5.38 5.40 5.39 5.40 0.19
亚油酸甲酯 0.42 0.42 0.42 0.42 0.42 0.42 0.42 0.48
亚麻酸甲酯 0.54 0.54 0.54 0.54 0.54 0.54 0.54 0.44









稳定性考察结果
脂肪酸甲酯 0h 2h 4h 6h 8h 平均 RSD(%)
正己酸甲酯 0.20 0.20 0.19 0.20 0.19 0.19 2.29
辛酸甲酯 0.41 0.41 0.39 0.41 0.41 0.41 2.01
癸酸甲酯 0.47 0.47 0.45 0.47 0.47 0.47 1.78
月桂酸甲酯 4.71 4.70 4.56 4.71 4.68 4.67 1.38
肉豆蔻酸甲酯 1.89 1.89 1.86 1.89 1.88 1.88 0.78
棕榈酸甲酯 1.76 1.76 1.76 1.77 1.76 1.76 0.17
棕榈油酸甲酯 0.03 0.03 0.03 0.03 0.03 0.03 0.51
硬脂酸甲酯 0.36 0.36 0.36 0.36 0.36 0.36 0.47
油酸甲酯 6.51 6.52 6.54 6.52 6.51 6.52 0.21
亚油酸甲酯 1.96 1.96 1.95 1.95 1.93 1.95 0.50
亚麻酸甲酯 0.15 0.15 0.15 0.15 0.15 0.15 0.71
根据对样品的预测结果，拟选择含量较高的饱和脂肪酸（月桂酸、肉豆蒄酸、棕榈酸）和不饱和脂肪酸（油酸、亚油酸）为测定指标订入质量标准，而方法学研究过程中将对USP35标准中的所有指标进行详细的方法学研究与验证，同时结合稳定性研究结果制定标准限度。
2.2 锯叶棕果实提取物软胶囊中β-谷甾醇含量测定实验方法和结果
2.2.1 分析方法
色谱条件：
仪器：高效液相色谱仪（岛津CBM-20A）
色谱柱：ALtima-C18（4.6×150mm，5μm）
流动相：乙腈：水，梯度洗脱，比例见下表。
流动相比例
时间 0.01 20 32 42 45 55
乙腈（%） 80 80 100 100 80 80
纯水（%） 20 20 0 0 20 20
检测波长：210nm
流速：1.0mL/min
进样量：20μL
柱温：30℃
稀释液：甲醇
空白溶液：稀释液
氢氧化钾溶液：取130g氢氧化钾置1000mL量瓶中，用200mL水溶解，加甲醇至刻度，摇匀。
对照品溶液：精确称定β-谷甾醇对照品1.5 mg，置1mL 量瓶中，用甲醇溶解并稀释至刻度，摇匀作为对照品溶液。
供试品溶液：取锯叶棕果实提取物软胶囊的内容物2.00g置100mL圆底烧瓶中，精密称定，加入20mL氢氧化钾溶液，100℃回流2小时，放冷，将提取液转移至150mL分液漏斗中，用水30mL洗涤圆底烧瓶，合并洗涤液。提取液用乙醚萃取3次，每次30mL，合并乙醚液。用水洗涤乙醚液3次，每次30mL。乙醚液蒸干，残渣甲醇溶解并转移至5mL量瓶中，即得供试品溶液。
2.2.2 实验结果

β-谷甾醇样品色谱图

β-谷甾醇75%对照品色谱图
β-谷甾醇样品和对照品分离结果
β-谷甾醇 保留时间（min） 拖尾因子 分离度 与相邻峰的分离度
样品 17.323 1.026 4.798 1.86
对照品 17.313 1.037 22.816 67.35
2.3 锯叶棕果实提取物软胶囊所需费用预算
Project Samples（shot） Time（day） COST（RMB，yuan）
Instruments Gas Chromatograph Methodology 1) Specificity； 3 1 6,600
2) Quantification limit, detection limit； 35 3
3) Standard curve； 18 2
4) Investigation of sample processing method； 36 4
5) Precision ；
Sampling precision； 6 1
Reproducibility； 12 2
Intermediate precision； 12 2
6) Recovery； 18 7
7) Stability； 5 1
8) Durability； 12 1
Content determination 1) Sample: 10 batches 40 3 1,500
2) Reference: (5+2 sampling) 21 2
Investigation of sample stability It needs long-term test, accelerating test and intermediate test for 15 times. Each time takes 20 shots for 2 days. 300 30 9,000
Liquid Chromatograph β-sitosterol Methodology (147 shots for 25 days) 1) Specificity； 3 1 7,500
2) Quantification limit, detection limit； 35 3
3) Standard curve: 18 shots for 2 days； 18 2
4) Investigation of sample processing method； 36 4
5) Precision
Sampling precision； 6 1
Reproducibility； 12 3
Intermediate precision； 12 3
6) Recovery； 18 7
7) Stability； 5 1
8) Durability； 12 2
Content determination Sample: 10 batches with 40 shots. 40 6 1,500
Reference: (5+2 sampling): 21 shots. 21
Investigation of sample stability It needs long-term test, accelerating test and intermediate test for 15 times. Each time takes 20 shots for 2 days. 300 30 9,000
Constant Temperature Heating Magnetic Stirrer It needs 6 stirrers for methyl esterification of fatty acid 3,600
Reagents 1)Acetonitrile 6 bottles (Tedia 4L), 1,620
3)Pure water 30 bottles (C’estbon) 88
4)N-hexane 1 bottle (4L) 500
5) Methanol: 1 bottle (4L) 110
6) Sulfuric acid 2 bottles (500mL) 60
7) Diethyl ether 15 bottles (500mL) 200
8)Sodium chloride 1 bottle (≥99.5%, 500g) 10
9)Potassium hydroxide 1 bottle (≥85%, 500g) 16
Standard Substances (I) Fatty acid methyl ester: methyl-n-caproate, methyl caprylate, methyl decanoate, methyl laurate, methyl myristate, methyl palmitate, methyl palmitoleate, methyl margarate, methyl stearate, methyl oleate, methyl linoleate and methyl linolenate. 1 Methodology and content determination 13,000
2 Sample stability: for long-term test, it has 0, 3, 6, 9, 12, 18, 24, 36 months; for accelerating test, it has 0, 3, 6 months; for intermediate stability test, it has 0, 6, 9, 12 months. It needs to be determined for 15 times 45,000
(II) β-sitosterol 1 Methodology and content determination: for 25mg/piece, it needs 8 pieces 23,000
2 Investigation of sample stability: for long-term test, it has 0, 3, 6, 9, 12, 18, 24, 36 months; for accelerating test, it has 0, 3, 6 months; for intermediate stability test, it has 0, 6, 9, 12 months. It needs to be determined for 15 times. For 25mg and piece, it needs 2 pieces, 92,000
Others 1）Organic filter (0.22μm): 400 pieces, 600
2）Glass syringe (1mL): 2 pieces, plastic syringe (1mL): 400 piece 250
3）Sampling vial: 100 pieces, 200
4） Volumetric flask: for 1mL, 20 pieces; for 2mL, 30 pieces; for 5mL, 30 pieces; for 10mL, 10 pieces; for 25mL, 5 pieces,; for 100mL, 3 pieces, 100
5）Eggplant-shaped flask: for 25mL, 6 pieces; for 100mL, 6 pieces, 180
6）Separating funnel: 125mL, 6 pieces, 570
7）Transfer pipette: for 1mL, 2 pieces; for 2mL, 2 pieces; for 5mL, 2 pieces, 100
Depreciation of equipment, administrative fee and cost for fuel and power 10,000
Experimenter’s salary 100,000
TOTAL 328,304

对照品价格
序号 名称 cas 编号 纯度 品牌 包装 单价 需要量 折后价
1 十二酸甲酯 111-82-0 234591-5G 99.50% (Aldrich) 5g 310.05 2 545.7
2 肉豆蔻酸甲酯 124-10-7 M3378 ≥99% (GC) Sigma 25g 786.24 1 691.9
3 棕榈酸甲酯 112-39-0 P5177 ≥99% (capillary GC) Sigma 5g 506.61 1 445.8
4 油酸甲酯 112-62-9 311111 99% Aldrich 5g 613.08 2 1079.0
5 亚油酸甲酯 112-63-0 L1876 ≥99% (GC) Sigma 5g 918.45 1 808.2
6 十七烷酸甲酯 1731-92-6 51633 analytical standard (Fluka) 5g 2,552.94 2 4493.2
7 己酸甲酯 106-70-7 21599 analytical standard Fluka 1ML-F 299.52 2 527.2
8 辛酸乙酯 106-32-1 112321 ≥99% Aldrich 5g 168.48 1 148.3
9 癸酸甲酯 110-42-9 299030 99% (Aldrich) 2.5g 525.33 1 462.3
10 硬脂酸甲酯 112-61-8 S5376 ~99% (GC) Sigma 5g 782.73 1 688.8
11 亚麻酸甲酯 301-00-8 L2626 ≥99% (GC) Sigma 1g 1396.98 2 2458.7
12 棕榈油酸甲酯 1120-25-8 P9667 ≥99% (capillary GC), liquid (Sigma) 500mg 876.33 1 771.2
13 β-谷甾醇 83-46-5 S1270 ≥95% Sigma 25mg 2895.75 8 23166
总价格 36286.3
以上是方法学验证时单次购买标准品需要的费用，考虑到对照品的稳定性，在进行预实验、方法学验证、样品限度制定、稳定性考察时分别购买对照品。预实验购买标准品两次，方法学验证、样品限度制定购买一次，稳定性考察分别进行0、3、6、9、12、18、24、36月样品检测，需要购买8次，一共需要购买11次。
A 脂肪酸甲酯对照品在方法学验证时购买量都如表格中所列，需要13000元，进行其它实验部分研究时只需要购买油酸甲酯、月桂酸甲酯、棕榈酸甲酯、肉豆蔻酸甲酯和亚油酸甲酯，需要45000元。
B β-谷甾醇对照品在方法学验证时购买量如表格所列需要23000元，其它实验研究部分每次需要数量为2，共需要92000元。
C预实验摸索条件需要购买以上对照品1批，其中β-谷甾醇需要4支，共计约23000元。
全部试验所需对照品一共需要18.6万元。
2.4 锯叶棕果实提取物软胶囊质量标准提高实验时间进度安排
The entire experiment is planned to be completed within 10 months:
Within the 1st month since this experiment starts, finish methodology validation for determining fatty acid;
In the 2nd month, finish determining fatty acid content for 10 batches of Sabal samples by gas chromatography and formulate content limitation;
In the 3rd month, finish methodology validation for determining β-sitosterol; 在锯叶棕果实提取物软胶囊质量标准提高中，选定的含量测定指标的限度确定：结合不同生产日期样品的测定结果（10批）及其中3批样品的稳定性试验结果确定含量限度，现在已经有相应的5个批号的样品

結果 (英文) 1:

Detection: chromatographic conditions as described above, and precise dosage is 0.5 µl sample for sample and standard solutions, content.2.1.2 resultFor sample color spectrumReference solution chromatogramChromatogram in the separation of components and the tailing factor is as follows:Fatty acid methyl esters in the control solution test solutionTrailing factor separated separation tailing factorMethyl caproate 32.00 0.97 24.31 0.96Methyl octanoate 40.01 1.00 36.30 1.00Capric acid methyl ester 45.90 1.04 45.02 1.01Methyl laurate 40.59 1.51 40.78 1.43Myristic acid methyl ester 32.88 1.17 33.55 1.13Palmitic acid methyl ester 30.71 1.21 31.29 1.15Palmitoleic acid methyl ester 4.54 1.13 4.54 1.0717-acid methyl ester of 10.82 1.09 11.16 1.09Methyl stearate 14.36 1.04 14.66 1.03Oleic acid methyl ester 3.55 1.65 3.59 1.68Linoleic acid methyl ester 6.61 1.16 6.60 1.14Linolenic acid methyl ester 9.71 1.02 9.75 1.03Results showed that reference Atlas and Atlas of test articles for each component meets the system requirements, and chromatographic Elution, lauric acid, meat and beans Guan acid, Palmitic acid, oleic acid and linoleic acid content is higher, lower other fatty acids.While for has control products solution of into sample precision test, results each component continuous into sample 6 times, component and within marked of response of than of RSD value maximum for 1.38%, for try products solution for has at room temperature stability investigation, results at room temperature placed 8h, each component and within marked of response of than RSD value maximum for 2.29% (flax acid methyl ester), basic meet determination requirements, data following table:Precision study resultsFatty acid methyl ester 1# 2# 3# 4# 5# 6# average RSD (%)Methyl caproate 1.14 1.14 1.14 1.13 1.14 1.14 1.14 0.26 Methyl octanoate 1.30 1.30 1.30 1.29 1.30 1.30 1.30 0.26 Capric acid methyl ester 13.01 12.99 12.97 12.92 13.01 12.97 12.98 0.25 Methyl laurate 5.22 5.22 5.21 5.20 5.22 5.21 5.21 0.17 Myristic acid methyl ester 4.87 4.87 4.86 4.85 4.87 4.86 4.86 0.15 Palmitic acid methyl ester 0.09 0.09 0.09 0.09 0.09 0.09 0.09 1.38 Palmitoleic acid methyl ester 2.76 2.76 2.75 2.75 2.76 2.76 2.76 0.14 Methyl stearate 17.97 17.98 17.93 17.93 17.98 17.97 17.96 0.13 Oleic acid methyl ester 5.41 5.41 5.39 5.38 5.40 5.39 5.40 0.19 Linoleic acid methyl ester 0.42 0.42 0.42 0.42 0.42 0.42 0.42-0.48 Linolenic acid methyl ester 0.54 0.54 0.54 0.54 0.54 0.54 0.54 0.44 Study on the stability of resultsFatty acid methyl ester 0h 2H 4H 6h 8h average RSD (%)Methyl caproate 0.20 0.20 0.19 0.20 0.19 0.19 2.29 Methyl octanoate 0.41 0.41 0.39-0.41 0.41 0.41 2.01 Capric acid methyl ester 0.47 0.47 0.45 0.47 0.47 0.47 1.78 Methyl laurate 4.71 4.70 4.56 4.71 to 4.68 4.67 1.38 Myristic acid methyl ester 1.89 1.89 1.86 1.89, 1.88 1.88 0.78 Palmitic acid methyl ester 1.76 1.76 1.76 1.77 1.76 1.76 0.17 Palmitoleic acid methyl ester 0.03 0.03 0.03 0.03 0.03 0.03 to 0.51 Methyl stearate 0.36 0.36 0.36 0.36 0.36 0.36 0.47 Oleic acid methyl ester 6.51 6.52 6.54 6.52 6.51 6.52 0.21 Linoleic acid methyl ester 1.96 1.96 1.95 1.95 1.93 1.95 0.50 Linolenic acid methyl ester 0.15 0.15 0.15 0.15 0.15 0.15 0.71 According to on samples of forecast results, intends select content high of saturated fatty acids (Laurel acid, and meat bean Guan acid, and Palm acid) and not saturated fatty acids (oil acid, and Asia oil acid) for determination indicators set into quality standard, and party law research process in the will on USP35 standard in the of all indicators for detailed of method learn research and validation, while combines stability research results developed standard limits.2.2 in saw palmetto fruit extract soft capsule of beta-sitosterol determination test method and results2.2.1 the analytical methodsChromatographic conditions:Instrumentation: high performance liquid chromatographic instrument (Shimadzu CBM-20A)Chromatographic column: ALtima-C18 (4.6x150mm,5 μ m)Mobile phase: acetonitrile: water, gradient Elution, the ratio shown in the table below.Mobile phaseTime 0.01 20 32 42 45 55Acetonitrile (%) 80 80 100 100 80 80Pure water (%) 20 20 0 0 20 20Detection wavelength: 210nmFlow rate: 1.0mL/minInjection volume: 20 μlColumn temperature: 30 cDiluent: methanolBlank solution: diluentPotassium hydroxide solution: apply 130g home 1000mL volumetric flasks, potassium hydroxide, 200mL water soluble, add methanol to scale, shake well.Reference solution: accurate 1.5 mg β-sitosterol reference, home 1mL, volumetric, dissolve and dilute with methanol to scale, shake the control solution.For the test solution: take saw palmetto fruit extract soft capsule contents 2.00g Chi 100mL in the round-bottomed flask, precision, 20mL potassium hydroxide solution and reflux for 2 hours at 100 ℃, cold, to transfer the extract to 150mL in a separatory funnel, water 30mL wash the round-bottom flask, merging the cleaning solution. Extract with ethyl ether 3 times per 30mL, the combined ether solution. Washing liquid ether 3 times per 30mL. Ether liquid evaporation to dryness, residues of methanol dissolved and transferred to 5mL, volumetric, for the test solution is obtained.2.2.2 resultsBeta-sitosterol sample chromatogramBeta-sitosterol 75% control color spectrum chartBeta-sitosterol separation of sample and reference resultsBeta-sitosterol retention time (min) trailing factor degrees of separation and the adjacent peaks of degrees of separationSample 17.323 4.798 1.86Control 17.313 22.816 67.352.3 saw palmetto fruit extract soft capsule costs budget Project Samples（shot） Time（day） COST（RMB，yuan）Instruments Gas Chromatograph Methodology 1) Specificity； 3 1 6,600 2) Quantification limit, detection limit； 35 3 3) Standard curve； 18 2 4) Investigation of sample processing method； 36 4 5) Precision ； Sampling precision； 6 1 Reproducibility； 12 2 Intermediate precision； 12 2 6) Recovery； 18 7 7) Stability； 5 1 8) Durability； 12 1 Content determination 1) Sample: 10 batches 40 3 1,500 2) Reference: (5+2 sampling) 21 2 Investigation of sample stability It needs long-term test, accelerating test and intermediate test for 15 times. Each time takes 20 shots for 2 days. 300 30 9,000Liquid Chromatograph β-sitosterol Methodology (147 shots for 25 days) 1) Specificity； 3 1 7,500 2) Quantification limit, detection limit； 35 3 3) Standard curve: 18 shots for 2 days； 18 2 4) Investigation of sample processing method； 36 4 5) Precision Sampling precision； 6 1 Reproducibility； 12 3 Intermediate precision； 12 3 6) Recovery； 18 7 7) Stability； 5 1 8) Durability； 12 2 Content determination Sample: 10 batches with 40 shots. 40 6 1,500Reference: (5+2 sampling): 21 shots. 21Investigation of sample stability It needs long-term test, accelerating test and intermediate test for 15 times. Each time takes 20 shots for 2 days. 300 30 9,000Constant Temperature Heating Magnetic Stirrer It needs 6 stirrers for methyl esterification of fatty acid 3,600 Reagents 1)Acetonitrile 6 bottles (Tedia 4L), 1,620 3)Pure water 30 bottles (C'estbon) 88 4)N-hexane 1 bottle (4L) 500 5) Methanol: 1 bottle (4L) 110 6) Sulfuric acid 2 bottles (500mL) 60 7) Diethyl ether 15 bottles (500mL) 200 8)Sodium chloride 1 bottle (≥99.5%, 500g) 10 9)Potassium hydroxide 1 bottle (≥85%, 500g) 16 Standard Substances (I) Fatty acid methyl ester: methyl-n-caproate, methyl caprylate, methyl decanoate, methyl laurate, methyl myristate, methyl palmitate, methyl palmitoleate, methyl margarate, methyl stearate, methyl oleate, methyl linoleate and methyl linolenate. 1 Methodology and content determination 13,0002 Sample stability: for long-term test, it has 0, 3, 6, 9, 12, 18, 24, 36 months; for accelerating test, it has 0, 3, 6 months; for intermediate stability test, it has 0, 6, 9, 12 months. It needs to be determined for 15 times 45,000(II) β-sitosterol 1 Methodology and content determination: for 25mg/piece, it needs 8 pieces 23,000 2 Investigation of sample stability: for long-term test, it has 0, 3, 6, 9, 12, 18, 24, 36 months; for accelerating test, it has 0, 3, 6 months; for intermediate stability test, it has 0, 6, 9, 12 months. It needs to be determined for 15 times. For 25mg and piece, it needs 2 pieces, 92,000Others 1）Organic filter (0.22μm): 400 pieces, 600 2）Glass syringe (1mL): 2 pieces, plastic syringe (1mL): 400 piece 250 3）Sampling vial: 100 pieces, 200 4） Volumetric flask: for 1mL, 20 pieces; for 2mL, 30 pieces; for 5mL, 30 pieces; for 10mL, 10 pieces; for 25mL, 5 pieces,; for 100mL, 3 pieces, 1005）Eggplant-shaped flask: for 25mL, 6 pieces; for 100mL, 6 pieces, 1806）Separating funnel: 125mL, 6 pieces, 570 7）Transfer pipette: for 1mL, 2 pieces; for 2mL, 2 pieces; for 5mL, 2 pieces, 100Depreciation of equipment, administrative fee and cost for fuel and power 10,000 Experimenter's salary 100,000 TOTAL 328,304 Reference priceSerial number purity brand name CAS number packaging price requirements for discounted price12 111-82-0 234591-5G 99.50% acid methyl ester (Aldrich) 5G 310.05 2 545.7 2 124-10-7 M3378 ≥ 99% myristic acid methyl ester (GC) Sigma 25G 786.24 1 691.9 3-methyl palmitate 112-39-0 P5177 ≥ 99% (capillary GC) Sigma 5G 506.61 445.8 4-methyl oleate 112-62-9 311111 99% Aldrich 5G 613.08 2 1079.0 5 112-63-0 L1876 ≥ 99% linoleic acid methyl ester (GC) Sigma 5G 918.45 1 808.2 Acid methyl ester 1731-92-6 67 51633 analytical standard (Fluka) 5G 2,552.94 2 4493.2 7-106-70-7 acid methyl ester 21599 analytical standard Fluka 1ML-F 299.52 2 527.2 8-ethyl 106-32-1 112321 ≥ 99% Aldrich 5G 1 148.3 110-42-9 299030 99% 9 decanoic acid methyl ester (Aldrich) 2.5G 525.33 1 462.3 10 stearic acid methyl ester 112-61-8 S5376 ~99% (GC) Sigma 5G 782.73 688.8 11 301-00-8 L2626 ≥ 99% linolenic acid methyl ester (GC) Sigma 1g 1396.98 2458.7 1120-25-8 P9667 ≥ 12 palmitoleic acid methyl ester 99% (capillary GC), liquid (Sigma) 500mg 876.33 1 771.2 13 beta-sitosterol ≥ 95% Sigma 83-46-5 S1270 25mg 2895.75 23166Total price 36286.3Above is what single methodology validation cost of purchasing standards require, taking into account the stability of the reference, in the preliminary experiments, validation method, formulation, stability when purchasing a sample limits reference standard. Pre-test purchase standard twice, validation method, samples limited to draw once, stability test with 0, 3, 6, 9, 12, 18, 24, 36 months samples tested, need to buy 8 times altogether to purchase 11 times.A reference substance of fatty acid methyl esters in the validation method when purchases are as listed in the table requires 13000 Yuan and other experimental studies only need to buy the oil acid methyl ester and methyl ester, Palmitic acid methyl ester of lauric acid and myristic acid methyl ester and linoleic acid methyl ester, need 45000 dollars.B β-sitosterol standard purchases when the validation method as listed in the table needs to 23000, other experimental studies section every time you need a quantity of 2 will require a total 92000.C test conditions may need to purchase more than 1 batch of reference standard, including beta-sitosterol takes 4, totalling about 23000.Reference substances required for full test need 186,000 yuan altogether.2.4 saw palmetto fruit extract soft capsule quality standards and time schedulesThe entire experiment is planned to be completed within 10 months:Within the 1st month since this experiment starts, finish methodology validation for determining fatty acid; In the 2nd month, finish determining fatty acid content for 10 batches of Sabal samples by gas chromatography and formulate content limitation; In the 3rd month, finish methodology validation for determining β-sitosterol; Saw Palmetto fruit extract soft capsule quality standards improve, determine the limits of the selected indicators for the determination of: combined results of samples with different production dates (10) and 3 samples of stability test results to determine concentration limits, now there is a corresponding 5 batches of samples

結果 (英文) 3:

Detection: according to the above chromatographic conditions, respectively precise amount of 0.5 L sample solution and the reference solution into the sample, content determination. The experimental results of
2.1.2



the chromatogram obtained from the test solution

control separation and tailing factor of each component solution chromatogram chromatogram are as follows:


fatty acid methyl ester standard solution sample solution
separation tailing factor separation tailing factor
methyl hexanoate 32 0.97 24.31 40.01 0.96
octanoic acid methyl ester 1 36.30 1.00
45.90 1.04 45.02 1.01
decanoic acid methyl ester methyl laurate 40.59 1.51 40.78 1.17 33 32.88 1.43
methyl myristate55 1.13
methyl palmitate 30.71 1.21 31.29 4.54 1.13 4.54 1.15
palmitic acid methyl ester 1.07
seventeen methyl 10.82 1.09 11.16 1.09
methyl stearate 14.36 1.04 14.66 3.55 1.65 3.59 1.68
1.03
fatty acid methyl ester methyl linoleate 6.61 1.16 6.60 1.14
linolenic acid methyl ester 9.71 1.02 9.75 1.03
the results show that: the standard spectrum and the tested product of each component accord with the system suitability requirements, at the same time from the sample spectra, lauric acid, myristic acid, palmitic acid, oleic acid and linoleic acid content is high, the other fatty acid content is low.
At the same time the reference solution sampling precision test, the results of each component of continuous sample 6, response components and the internal standard as the ratio of the maximum RSD value is 1.38%, the test solution was investigated at room temperature stability at room temperature, the results of 8h, the components and the ratio of the response of the internal standard RSD value a maximum of 2.29% (linolenic acid methyl ester), in line with the basic requirements for the determination, data are listed in the following table:




precision inspection results of fatty acid methyl ester 1 2 3 4 5 6 average RSD (%)
methyl hexanoate 1.14 1.14 1.14 1.13 1.14 1.14 1.14 0.26 1.30 1.30 1.30 1.29
octanoic acid ethyl ester 1301.30 1.30 0.26 13.01 12.99 12.97
decanoic acid methyl ester 12.92 13.01 12.97 12.98 0.25 5.22 5.22
lauric acid methyl ester 5.21 5.20 5.22 5.21 5.21 0.17
methyl myristate 4.87 4.87 4.86 4.85 4.87 4.86 4.86 0.15 0.09 0.09
palmitic acid methyl ester 0.09 0.09 0.09 0.09 0.09 1.38 2.76 2.76 2.75
palmitic acid methyl ester 2.75 2.76 2.76 2.76 0.14
methyl stearate, 17.97 17.98 17.93 17.93 17.98 17.97 17.96 0.13
Methyl oleate 5.41 5.41 5.39 5.38 5.40 5.39 5.40 0.19 0.42 0.42
linoleic acid methyl ester 0.42 0.42 0.42 0.42 0.42 0.48
linolenic acid methyl ester 0.54 0.54 0.54 0.54 0.54 0.54 0.54 0.44









stability investigation results
fatty acid methyl ester 0h 2H 4H 6h 8h the mean RSD (%)
methyl hexanoate 0.20 0.20 0.19 0.20 0.19 0.19 2.29 0.41 0.41 0.39
octanoic acid methyl ester 0.41 0.41 0.41 2.01 0.47 0.47 0.45
decanoic acid methyl ester 0.47 0.47 1.78
0.47Lauric acid methyl ester 4.71 4.70 4.56 4.71 4.68 4.67 1.38
methyl myristate 1.89 1.89 1.86 1.89 1.88 1.88 0.78 1.76 1.76
palmitic acid methyl ester 1.76 1.77 1.76 1.76 0.17 0.03 0.03 0.03
palmitic acid methyl ester 0.03 0.03 0.03 0.51 0.36 0.36
methyl stearate 0.36 0.36 0.36 0.36 0.47 6.51 6.52 6.54
methyl oleate 6.52 6.51 6.52 0.21
linoleic acid methyl ester 1.96 1.96 1.95 1.95 1.93 1.95 0.50 0.15 0.15 0
linolenic acid methyl ester150.15 0.15 0.15 0.71
according to the prediction results of samples, intends to choose high saturated fat content acid (lauric acid, myristic acid, palmitic acid) and unsaturated fatty acids (oleic acid, linoleic acid) for the determination of indicators established quality standard, and the research methodology will process all indexes in USP35 standard detailed methodological research and verification, combined with the results of the stability study of the formulation of a standard limit. The experimental method and results of the determination of the content of beta sitosterol palmetto fruit extract soft capsule in the
2.2.1 analysis method of chromatographic conditions:


2.2 saw instrument: high performance liquid chromatograph (SHIMADZU CBM-20A)
chromatographic column:ALtima-C18 (4.6 x 150mm, 5 m)
: acetonitrile: water mobile phase, gradient elution ratio, see the following table.

0.0120324245 55
proportion of mobile phase acetonitrile (%) of 808010010080 80
pure water (%) 20200020 20
detection wavelength: 210nm
flow rate: 1.0mL/min
sample size: 20 L
column temperature: 30 C

blank solution dilution: methanol: dilution
KOH solution: take 130g potassium hydroxide into 1000mL volumetric flask, dissolved 200mL water, add methanol to the scale, shake.
control solution: accurate said beta sitosterol control article 1.5 mg, into 1mL volumetric flask,Dissolved in methanol and diluted to the scale, shake as the reference solution.
solution for test: taking saw palmetto fruit extract soft capsule content 2.00g the 100mL round bottom flask, accurately weighed, to join the 20mL potassium hydroxide solution, the temperature of 100 DEG C to reflux for 2 hours, cool, the extract was transferred to a separatory funnel in 150mL, 30mL water washing round bottom flask, and washing liquid. Ether extraction extract 3 times, each time 30mL, combined ether liquid. 3 times of water washing ether solution, each 30mL. The ether solution is evaporated to dryness, the residue dissolved in methanol and transferred to a 5mL volumetric flask, to obtain the solution for test. The experimental results of
2.2.2

Beta sitosterol sample chromatogram

- sitosterol 75% control chromatograms of
- sitosterol sample and the control results fractions
- sitosterol retention time (min) tailing factor and the degree of separation of adjacent peak separation degree
17.323 1.026 4.798 1.86
sample control products 17.313 1.037 22.816 67.35
2.3 saw palmetto fruit extract soft capsule required cost budget of the
Project Samples (shot) Time (day) COST (RMB, yuan)
Instruments Gas Chromatograph Methodology 1) Specificity 316600
2); Quantification limit,Detection limit; Standard curve; 3) 353
182
4) Investigation of sample processing method; Precision; 364
5)
61
Reproducibility Sampling precision; Intermediate precision; 122
122
6); Recovery; Stability; 51 187
7)
8)
121 Durability; Content determination 1) Sample: 10 batches 4031500
2)
212 Reference: (52 sampling)Investigation of sample stability It needs long-term test, accelerating test and intermediate test for 15 times. Each time takes 20 shots for 2 days. 300309000
Liquid Chromatograph beta -sitosterol Methodology (147 shots for 25 days 1 Specificity 317500;))
2) Quantification limit, detection limit; 353
3) Standard curve: 18 shots for 2 days 182

;4) Investigation of sample processing method; 364
5)
61
Precision Sampling precision; Reproducibility; Intermediate precision 123
123
6); Recovery; Stability; 7) 187
51
8)
122 Durability; Content determination Sample: 10 batches with 40 shots. 4061500
Reference: (52 sampling): 21 shots. 21
Investigation of sample stability It needs long-term test, accelerating test and intermediate test for 15 times. Each time takes 20 shots for 2 days. 300309000 Temperature Heating Magnetic Stirrer
Constant It needs 6 stirrers for methyl esterification of fatty acid 3600
Reagents 1) Acetonitrile 6 bottles (Tedia 4L), 1620
3 Pure water 30 bottles (C) '88
estbon)4) N-hexane 1 bottle (4L) 500
5) Methanol: 1 bottle (4L) 110
6) Sulfuric acid 2 bottles (500mL) 60
7) Diethyl ether 15 bottles (500mL) 200
8) Sodium chloride 1 (bottle = 99.5%, 500g) 10
9) Potassium hydroxide 1 (= bottle 85%, 500g 16)
Standard Substances (I) Fatty acid methyl ester: methyl-n-caproate, methyl caprylate, methyl decanoate,Methyl laurate, methyl myristate, methyl palmitate, methyl palmitoleate, methyl margarate, methyl stearate, methyl oleate, methyl linoleate and methyl linolenate. 1 Methodology and content determination Sample stability: for 13000
2 long-term test, it has 0, 3, 6, 9, 12, 18, 24, 36 months; for accelerating test, it has 0, 3, 6 months; for intermediate stability test, it has 0, 6, 9,12 months. It needs to be determined for 15 times 45000
(II) Methodology and content 1 beta -sitosterol determination: for 25mg/piece, it needs 8 pieces 23000 of sample stability:
2 Investigation for long-term test, it has 0, 3, 6, 9, 12, 18, 24, 36 months; for accelerating test, it has 0, 3, 6 months; for intermediate stability test, it has 0, 6, 9, 12 months.