A quality detection method of shen-shuai-kang capsules

Abstract

The application discloses a quality control method of Shen Shuai Kang capsules, and applies isoxanthohumol to the quality control of the Shen Shuai Kang capsules, carries out thin-layer identification and fingerprint spectrum detection on the Shen Shuai Kang capsules, and determines the content of isoxanthohumol in the Shen Shuai Kang capsules, so that the quality control standard of the Shen Shuai Kang capsules is improved. The method can not only more accurately, comprehensively and conveniently evaluate the quality of the Shen Shuai Kang capsules, but also indirectly guarantee the effectiveness and safety of the product. The quality control method has the advantages of simple sample preparation method, reliable and stable detection result and the like.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to the technical field of drug detection, and particularly relates to a quality detection method of Shen Shuai Kang capsules. BACKGROUND

[0002] Shen Shuai Kang capsules have the effects of tonifying the body resistance, benefiting the intelligence, soothing the nerves and invigorating the kidney and spleen, and are used for treating the symptoms of kidney and spleen yang deficiency, soreness of the waist and knees, physical weakness, insomnia, dreaminess and loss of appetite. The main component of Shen Shuai Kang capsules is the dried tender stems of Acanthopanax henryi, which is prepared by water extraction.

[0003] In order to ensure the quality of drugs, quality control of drugs needs to be performed during the production of drugs. At present, the quality detection method of Shen Shuai Kang capsules is recorded in the “Ministry of Health Drug Standards·Traditional Chinese Medicine Prescriptions” issued by the Ministry of Health in 1997, the 12th volume, page 147. The quality control of Shen Shuai Kang capsules mainly includes two items: one is to identify whether there are plant sugar and phenolic compounds in Shen Shuai Kang capsules through a physicochemical color reaction; and the other is to detect the content of rutin by preparing a standard curve and reading the weight of rutin in the test solution of Shen Shuai Kang capsules. For the physicochemical color reaction, since all the medicinal materials derived from higher plants contain similar compounds of sugar and phenolic compounds, the identification method has poor specificity, cannot prove that Acanthopanax henryi is used in the prescription, and cannot effectively evaluate the quality and authenticity of Shen Shuai Kang capsules. When detecting the content of rutin, the operation is relatively complicated, the standard curve needs to be prepared repeatedly, the linearity is not good, the detection result changes greatly, and the sodium nitrite-aluminum nitrate reaction has many influencing factors, the human error of the operator is large, the reproducibility and precision of the detection result do not meet the requirements. Therefore, there is a lack of quality detection method for Shen Shuai Kang capsules at present, which brings great risks to the controllability of the quality of Shen Shuai Kang capsules, and even directly affects the medicinal material basis, effectiveness and safety of the drug, which does not meet the requirements of the current “People's Republic of China Pharmacopoeia (2020 Edition)”.

[0004] Therefore, it is urgent to develop a quality detection method for Shen Shuai Kang capsules. SUMMARY

[0005] In view of this, the present disclosure provides a quality detection method of Shen Shuai Kang capsules to make up for the defects in the existing quality control standard of Shen Shuai Kang capsules.

[0006] The technical scheme provided by the present disclosure is specifically a quality detection method of Shen Shuai Kang capsules, and the control method includes one or more detections of the Shen Shuai Kang capsules as follows:

[0007] 1) Thin-layer identification: identifying whether there is isofraxidin in the Shen Shuai Kang capsules;

[0008] 2) Fingerprint detection: determining the content of isofraxidin in the Shifa Kang capsule and / or detecting the fingerprint of the Shifa Kang capsule.

[0009] Preferably, the specific process of the thin layer identification is as follows:

[0010] Test solution: Take the fine powder of the Shifa Kang capsule, add methanol, ultrasonic extraction, filter, evaporate the filtrate to dryness, dissolve with water, extract the water layer with chloroform for multiple times, combine the chloroform layers, evaporate to dryness, dissolve the residue with methanol to obtain the test solution;

[0011] Controlled drug solution: Take the controlled drug of Acanthopanax brachystachyus, add methanol, ultrasonic extraction, filter, evaporate the filtrate to dryness, dissolve with water, extract the water layer with chloroform for multiple times, combine the chloroform layers, evaporate to dryness, dissolve the residue with methanol to obtain the controlled drug solution;

[0012] Controlled product solution: Take the controlled product of isofraxidin, dissolve with methanol to obtain the controlled product solution of isofraxidin;

[0013] Thin layer chromatography identification: take the test solution, the controlled drug solution and the controlled product solution, respectively, and point them on the thin layer plate, add the developing agent, develop, take out, dry, fumigate with ammonia, and observe under ultraviolet light at 365 nm, identify the same color fluorescent spots in the test chromatogram, the controlled product chromatogram and the controlled drug chromatogram of Acanthopanax brachystachyus.

[0014] Further preferably, the preparation of the test solution is as follows:

[0015] Take 1.0 g of the fine powder of the Shifa Kang capsule, add 35 ml of methanol, ultrasonic extraction for 30 min, filter, evaporate the filtrate to dryness, dissolve the residue with 15 ml of water, extract the water layer with chloroform for 3 times, each time 10 ml, combine the chloroform layers, evaporate to dryness, dissolve the residue with 1 ml of methanol to obtain the test solution;

[0016] The controlled drug solution is as follows:

[0017] Take 2.0 g of the controlled drug of Acanthopanax brachystachyus, add 35 ml of methanol, ultrasonic extraction for 30 min, filter, evaporate the filtrate to dryness, dissolve the residue with 15 ml of water, extract the water layer with chloroform for 3 times, each time 10 ml, combine the chloroform layers, evaporate to dryness, dissolve the residue with 1 ml of methanol to obtain the controlled drug solution.

[0018] Further preferably, the concentration of isofraxidin in the controlled product solution is 0.5 mg / ml.

[0019] Further preferably, in the thin layer chromatography identification, the amount of the test solution is 5-10 μl, the amount of the controlled drug solution is 5 μl, and the amount of the controlled product solution is 3 μl.

[0020] Further preferably, in the thin layer chromatography identification, a mixed solution of toluene, ethyl acetate and formic acid is used as the developing agent, wherein the toluene: ethyl acetate: formic acid = (25-35): (5-8): (1-3) by volume.

[0021] Further preferably, the content of isofraxidin in the Shen Shua Kang capsule is: not less than 0.3 mg of isofraxidin per 1 Shen Shua Kang capsule calculated on the dry basis.

[0022] Further preferably, the fingerprint detection is specifically:

[0023] 1) Preparation of test sample: 1.0 g of sample was weighed, 35 ml of methanol was added, and ultrasonic was performed for 30 min. The filtrate was evaporated to dryness, and the residue was dissolved in 5 ml of methanol. The solution was filtered through a 0.45 μm membrane.

[0024] 2) Preparation of control solution: isofraxidin control was weighed, and a solution containing 15 μg of isofraxidin per 1 ml was prepared by adding 60% methanol by volume;

[0025] 3) Octadecylsilane-bonded silica gel was used as the filler: Phenomenex Luna, column length 250 mm, column inner diameter 4.6 mm, particle size 5 μm; methanol was used as the mobile phase A, and 0.1% phosphoric acid solution was used as the mobile phase B, which was eluted by gradient; the flow rate was 1 ml per minute; the detection wavelength was 344 nm; the column temperature was 30°C;

[0026] 4) Determination: 10 μl of the control solution and the test sample solution were precisely taken and injected into the liquid chromatograph, respectively, to obtain the fingerprint and determine the content of isofraxidin.

[0027] Further preferably, the gradient elution conditions in step 3) are specifically: 0-35 min, 10% A; 35-55 min, 10-30% A; 55-85 min, 30-50% A; 85-100 min, 50-70% A; 100-105 min, 70-10% A; 105-115 min, 10% A.

[0028] Further preferably, the main chemical components of the Shen Shua Kang capsule in the fingerprint detection are specifically:

[0029] The fingerprint has 14 common peaks, and the retention time of each common peak is: peak 1: relative retention time 7.819; peak 2: relative retention time 13.495; peak 3: relative retention time 23.411; peak 4: relative retention time 37.051; peak 5: relative retention time 39.112; peak 6: relative retention time 43.519; peak 7: relative retention time 50.398; peak 8: relative retention time 53.594; peak 9: relative retention time 55.622; peak 10: relative retention time 68.107; peak 11: relative retention time 71.349; peak 12: relative retention time 78.294; peak 13: relative retention time 93.075; and peak 14: relative retention time 107.652.

[0030] The quality detection method of Shen Shuai Kang capsules provided by the application applies isoxypinidine to the quality control of Shen Shuai Kang capsules, and determines whether isoxypinidine exists in the Shen Shuai Kang capsules and the content of isoxypinidine through thin layer identification and fingerprint detection of the Shen Shuai Kang capsules, so as to verify the existence and content of the effective components in Acanthopanax brachystachyus, and ensure the quality of the Shen Shuai Kang capsules. The method can not only more accurately, comprehensively and conveniently evaluate the quality of the Shen Shuai Kang capsules, but also indirectly guarantee the effectiveness and safety of the product.

[0031] The quality detection method of Shen Shuai Kang capsules provided by the application has the advantages of simple test sample processing and reliable and stable detection results.

[0032] It should be understood that the foregoing general description and the following detailed description are only exemplary and explanatory, and cannot limit the disclosure of the application. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 It is a fluorescence spot comparison chart of the test sample chromatogram, the isoxypinidine reference chromatogram and the Acanthopanax brachystachyus reference medicinal material chromatogram in the disclosed embodiment 1 of the application, wherein 1 is isoxypinidine, 2 is a reference medicinal material, and 3 is a control drug.

[0034] Figure 2 It is a fluorescence spot comparison chart of the test sample chromatogram, the isoxypinidine reference chromatogram and the Acanthopanax brachystachyus reference medicinal material chromatogram in the disclosed embodiment 2 of the application, wherein 1 is isoxypinidine, 2 is a reference medicinal material, 3 is a batch of Shen Shuai Kang capsules, 4 is another batch of Shen Shuai Kang capsules, and 5 is a third batch of Shen Shuai Kang capsules.

[0035] Figure 3 It is a fluorescence spot comparison chart of the test sample chromatogram, the isoxypinidine reference chromatogram and the Acanthopanax brachystachyus reference medicinal material chromatogram in the disclosed embodiment 3 of the application, wherein 1 is isoxypinidine, 2 is a reference medicinal material, 3 is a fourth batch of Shen Shuai Kang capsules, 4 is a fifth batch of Shen Shuai Kang capsules, and 5 is a sixth batch of Shen Shuai Kang capsules.

[0036] Figure 4 The fingerprint spectrum detected in Example 4 of the present application discloses Shen Shuai Kang Capsules;

[0037] Figure 5 The fingerprint spectrum detected in 11 batches of Shen Shuai Kang Capsules in the present application is disclosed. DETAILED DESCRIPTION

[0038] The present application will be further explained in combination with specific embodiments, but is not used to limit the protection scope of the present application.

[0039] In view of the fact that the existing standard cannot meet the quality standard of Shen Shuai Kang Capsules, the present inventors have found, after a large amount of research, that in the past, when detecting S. involvulus, the detection was mostly based on the calibration component of syringin, while in Shen Shuai Kang Capsules, isofraxidin is the core effective component of S. involvulus. Therefore, for the first time, the detection of S. involvulus is attempted based on isofraxidin as the calibration material, so as to realize the quality control of Shen Shuai Kang Capsules.

[0040] In order to realize the identification and content determination of isofraxidin in Shen Shuai Kang Capsules, thin layer identification and fingerprint spectrum detection are adopted in the present embodiment. Since the thin layer identification has the characteristics of clear spots, strong specificity and high stability, it is conducive to controlling the internal quality of Shen Shuai Kang Capsules. The method of content determination by fingerprint spectrum has the advantages of high precision, good reproducibility and strong stability, and meets the relevant requirements of Chinese Pharmacopoeia Appendix.

[0041] The thin layer identification and fingerprint spectrum detection in the above-mentioned embodiments will be further described in detail in combination with specific embodiments.

[0042] Example 1: Thin layer identification

[0043] S1: Preparation of test solution: take 1 g of Shen Shuai Kang Capsules, add 35 ml of methanol, ultrasonic for 0.5 h, filter, evaporate to dryness, add 15 ml of water to dissolve, extract with chloroform for 3 times, 10 ml each time, take the chloroform layer, evaporate to dryness, dissolve the residue with 1 ml of methanol as the test solution.

[0044] S2: Preparation of control drug solution: take 2 g of S. involvulus control drug, add 35 ml of methanol, ultrasonic for 0.5 h, filter, evaporate to dryness, add 15 ml of water to dissolve, extract with chloroform for 3 times, 10 ml each time, take the chloroform layer, evaporate to dryness, dissolve the residue with 1 ml of methanol as the control drug solution.

[0045] S3: Take isofraxidin, add methanol to prepare a control solution of 0.5 mg / ml.

[0046] S4: Thin-layer identification: 2-5ul of each point on the same silica gel G plate, with toluene-ethyl acetate-formic acid = 31:8:1 as developing agent to develop about 10 cm, take out, dry, ammonia fumigation, and then observe under UV 365nm. In the test sample chromatogram, the same color fluorescent spots appeared at the corresponding positions of the control sample chromatogram, and the positive sample corresponded well. See Figure 1 .

[0047] Example 2: Thin-layer identification

[0048] S1: Preparation of test sample solution: Take 1g of each of the 1st, 2nd and 3rd batches of Shen Shuaikang capsules, add 35ml of methanol and ultrasonic for 0.5h, filter, evaporate to dryness, add 15ml of water to dissolve the residue, extract with chloroform 3 times, 10ml each time, take the chloroform layer, evaporate to dryness, and dissolve the residue with 1ml of methanol as the test sample solution.

[0049] S2: Preparation of control medicinal material solution: Take 2g of the control medicinal material of Acanthopanax negundo var. sinicus, add 35ml of methanol and ultrasonic for 0.5h, filter, evaporate to dryness, add 15ml of water to dissolve the residue, extract with chloroform 3 times, 10ml each time, take the chloroform layer, evaporate to dryness, and dissolve the residue with 1ml of methanol as the control medicinal material solution.

[0050] S3: Take isoxazoline skin and add methanol to prepare a 0.5mg / ml control sample solution.

[0051] S4: Thin-layer identification: 2-5ul of each point on the same silica gel G plate, with toluene-ethyl acetate-formic acid = 25:5:1 as developing agent to develop about 10 cm, take out, dry, ammonia fumigation, and then observe under UV 365nm. In the test sample chromatogram, the same color fluorescent spots appeared at the corresponding positions of the control sample chromatogram, and the positive sample corresponded well. See Figure 2 .

[0052] Example 3: Thin-layer identification

[0053] S1: Preparation of test sample solution: Take 1g of each of the 1st, 2nd and 3rd batches of Shen Shuaikang capsules, add 35ml of methanol and ultrasonic for 0.5h, filter, evaporate to dryness, add 15ml of water to dissolve the residue, extract with chloroform 3 times, 10ml each time, take the chloroform layer, evaporate to dryness, and dissolve the residue with 1ml of methanol as the test sample solution.

[0054] S2: Preparation of control medicinal material solution: Take 2g of the control medicinal material of Acanthopanax negundo var. sinicus, add 35ml of methanol and ultrasonic for 0.5h, filter, evaporate to dryness, add 15ml of water to dissolve the residue, extract with chloroform 3 times, 10ml each time, take the chloroform layer, evaporate to dryness, and dissolve the residue with 1ml of methanol as the control medicinal material solution.

[0055] S3: Take isoxazoline skin and add methanol to prepare a 0.5mg / ml control sample solution.

[0056] S4: Thin layer identification: 2-5ul of each point on the same silica gel G plate, with toluene-ethyl acetate-formic acid=35:8:3 as developing agent, developed for about 10 cm, taken out, dried, ammonia fumigation, and observed under UV 365nm. The test chromatogram showed the same color fluorescent spots at the corresponding positions of the control chromatogram, and the positive sample corresponded well. See Figure 3 .

[0057] Example 4: Fingerprint detection

[0058] 1) Preparation of test sample

[0059] Take 1.0g of sample, accurately weigh, ultrasonic for 30min with 35ml of methanol, filter, evaporate the filtrate to dryness, add methanol to 5ml; filter through 0.45μm membrane;

[0060] 2) Preparation of control solution Respectively take isoxazole piperidine control product, accurately weigh, add 60% methanol to prepare a solution containing 15μg of isoxazole piperidine per 1ml;

[0061] 3) Octadecylsilane-bonded silica gel as filler (Phenomenex Luna, column length 250mm, column inner diameter 4.6mm, particle size 5μm); methanol as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, gradient elution according to the provisions in 5); flow rate is 1ml per minute; detection wavelength is 344nm; column temperature is 30°C. The theoretical plate number calculated by isoxazole piperidine peak should not be less than 6000.

[0062] 4) Determination method: accurately pipette 10μl of control solution and test sample solution respectively, inject into liquid chromatograph, and determine; calculated by dry product, each 1g contains not less than 30ug of isoxazole piperidine.

[0063] 5) Gradient elution conditions as follows: 0-35min, 10%A; 35-55min, 10-30%A; 55-85min, 30-50%A; 85-100min, 50-70% A; 100-105min, 70-10%A; 105-115min, 10%A.

[0064] Among them, the obtained Shen Shuai Kang capsule fingerprint spectrum is as follows: Figure 4 .

[0065] Example 5: Fingerprint detection

[0066] 1) Precision test

[0067] The precision of 10 μl of the same sample solution was measured by repeating the injection 6 times, and the peak area of isofraxidin was calculated. The RSD of the peak area was 0.09%, indicating that the precision of the instrument was good. The RSD of the retention time and peak area of each target peak was less than 2, and the precision of the instrument was good.

[0068] Table 1. Precision test results (n = 6)

[0069]

[0070] 2) Stability test

[0071] The precision of 10 μl of the same sample solution was measured by repeating the injection 6 times, and the peak area of isofraxidin was calculated. The RSD of the peak area was 0.75% at 0, 2, 4, 8, 12, 18 and 24 h, respectively. The RSD of the retention time and peak area of each target peak was less than 2, and the results showed that the sample solution was stable within 24 h.

[0072] Table 2. Stability test results (n = 7)

[0073]

[0074] 3) Reproducibility test

[0075] Six samples of the same batch (batch number 220103) were extracted according to the sample solution preparation method and analyzed according to the proposed chromatographic conditions. The total amount of isofraxidin was 200.12 ug / g, and the RSD was 0.41%. The RSD of the retention time and peak area of each target peak was less than 2, and the results showed that the reproducibility of the method was good.

[0076] Table 3. Reproducibility test results (n = 6)

[0077]

[0078] 4. Sample addition recovery rate Six samples of known content (batch number 220103), each weighing about 0.50 g, were precisely weighed, and isofraxidin reference substance solution was precisely added to the samples. The sample solution was prepared according to the sample solution preparation method, and the chromatographic conditions were determined. The recovery rate of isofraxidin was calculated. The results are shown in the table. The results showed that the recovery rate of isofraxidin was between 95% and 105%, indicating that the method had good recovery rate.

[0079] Table 4. Isofraxidin sample addition recovery results (n = 6)

[0080]

[0081] According to the determination results of 11 batches of Shen Shuai Kang capsules, see Figure 5 , calculated as dry product, each Shen Shuai Kang capsule contains not less than 0.3 mg of isofraxidin.

[0082] Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope and spirit of the application being indicated by the following claims.

[0083] It is to be understood that the application is not limited to particular details described herein and that various modifications and changes can be made to the embodiments described without departing from the scope of the application. It is intended that the specification and depicted embodiments be considered as exemplary only, with the true scope and spirit of the application being indicated by the following claims.

Claims

1. A quality testing method for Shenshuikang capsules, characterized in that: 1) Thin-layer chromatography identification: to identify isozymine in the Shenshuikang capsules; Test solution: Weigh the fine powder of Shenshuikang capsules, add methanol, extract by ultrasonication, filter, evaporate the filtrate to dryness, add water to dissolve, extract the aqueous layer with chloroform, combine the chloroform layers, evaporate to dryness, dissolve the residue in methanol to obtain the test solution; Reference herb solution: Take the reference herb Acanthopanax senticosus, add methanol, extract by ultrasonication, filter, evaporate the filtrate to dryness, add water to dissolve, extract the aqueous layer with chloroform multiple times, combine the chloroform layers, evaporate to dryness, dissolve the residue in methanol to obtain the reference herb solution; Reference solution: Weigh out isozinetidine reference standard, dissolve in methanol to prepare isozinetidine reference solution; Thin-layer chromatography identification: Take the test solution, reference medicinal material solution and reference solution and spot them on a thin-layer plate respectively. Add toluene, ethyl acetate and formic acid in a volume ratio of (25-35):(5-8):(1-3). Develop, remove, air dry, fumigate with ammonia and examine under ultraviolet light at 365 nm. In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatograms of isozymine reference standard and obovate leaf ginseng reference medicinal material. 2) Fingerprint spectrum detection: Determine the content of isozymine in the Shenshuikang capsules and the similarity of the fingerprint spectrum of the Shenshuikang capsules; Test solution: Weigh 1.0 g of sample, add 35 ml of methanol, sonicate for 30 min, filter, evaporate to dryness, add methanol to the residue to make up to 5 ml, and filter through a 0.45 μm membrane; Reference solution: Weigh out isopyridine reference standard and add 60% methanol to prepare a solution containing 15 μg of isopyridine per 1 ml; The column was filled with octadecylsilane-bonded silica gel (Phenomenex Luna), with a column length of 250 mm, an inner diameter of 4.6 mm, and a particle size of 5 μm. Methanol was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B. Gradient elution was performed, with the following conditions: 0-35 min, 10% A; 35-55 min, 10-30% A; 55-85 min, 30-50% A; 85-100 min, 50-70% A; 100-105 min, 70-10% A; 105-115 min, 10% A; flow rate of 1 ml / min; detection wavelength of 344 nm; and column temperature of 30 °C. Determination: Accurately pipette 10 μl of the reference solution and the test solution into the liquid chromatograph to obtain fingerprint chromatograms, and simultaneously determine the content of isozymine.

2. The quality testing method for Shenshuikang capsules according to claim 1, characterized in that, In the thin-layer identification, 1.0g of Shenshuikang capsule fine powder was weighed, 35ml of methanol was added, and ultrasonic extraction was performed for 30min. The mixture was filtered, the filtrate was evaporated to dryness, and the residue was dissolved in 15ml of water. The aqueous layer was extracted three times with 10ml of chloroform each time. The chloroform layers were combined, evaporated to dryness, and the residue was dissolved in 1ml of methanol to obtain the test solution. Weigh 2.0g of the reference herb *Acanthopanax senticosus*, add 35ml of methanol, extract by sonication for 30min, filter, evaporate the filtrate to dryness, dissolve the residue in 15ml of water, extract the aqueous layer three times with 10ml of chloroform each time, combine the chloroform layers, evaporate to dryness, dissolve the residue in 1ml of methanol, and use this as the reference herb solution.

3. The quality testing method for Shenshuikang capsules according to claim 1, characterized in that, In the thin-layer chromatography identification, the concentration of isozypicoride in the reference solution is 0.5 mg / ml.

4. The quality testing method for Shenshuikang capsules according to claim 1, characterized in that, In the thin-layer chromatography identification, the amount of the test sample solution is 5-10 μl, the amount of the reference medicinal material solution is 5 μl, and the amount of the reference standard solution is 3 μl.

5. The quality testing method for Shenshuikang capsules according to claim 1, characterized in that, The isozyzine content in the Shenshuikang capsules, calculated on a dried basis, is no less than 0.3 mg per capsule.

6. The quality testing method for Shenshuikang capsules according to claim 1, characterized in that, The specific requirements for the main chemical components of Shenshuikang capsules in the fingerprint spectrum detection are as follows: The fingerprint spectrum contains 14 common peaks, with the following retention times: Peak 1: relative retention time 7.819; Peak 2—isoazine-corticoline: relative retention time 13.495; Peak 3: relative retention time 23.411; Peak 4: relative retention time 37.051; Peak 5: relative retention time 39.112; Peak 6: relative retention time 43.519; Peak 7: relative retention time 50.398; Peak 8: relative retention time 53.594; Peak 9: relative retention time 55.622; Peak 10: relative retention time 68.107; Peak 11: relative retention time 71.349; Peak 12: relative retention time 78.294; Peak 13: relative retention time 93.075; Peak 14: relative retention time 107.652.

Images