Liquid chromatography method for rapidly determining D / L-amygdalin in traditional Chinese medicine semen armeniacae amarae

Abstract

The invention discloses a liquid chromatography method for rapidly determining D / L-amygdalin in traditional Chinese medicine semen armeniacae amarae / semen persicae. A chromatographic column stationary phase used in the method is a silica gel matrix bonded biphenyl reversed-phase chromatography filler; wherein the type of the silica gel substrate comprises full-porous silica gel, core-shell surface porous silica gel and hybrid silica gel; the bonded chemical functional group is a biphenyl group. According to the method, silica gel substrate bonded biphenyl reversed-phase chromatographic packing is adopted as a chromatographic column stationary phase, and optimal chromatographic column temperature, mobile phase type, flow velocity, elution method and the like are combined, so that the D / L-amygdalin in the traditional Chinese medicine semen armeniacae amarae / semen persicae is rapidly measured; according to the present invention, with the method, the baseline separation of the D / L-amygdalin in the sample can be achieved within the short analysis time, the good separation selectivity on the two epimers of the D / L-amygdalin is provided, the universality is provided, and the method can be simultaneously suitable for the determination of the D / L-amygdalin in the traditional Chinese medicine semen armeniacae amarae and the semen persicae.

Description

Technical Field

[0001] The invention relates to the field of chemical analysis, in particular to a liquid chromatography method for rapidly determining D / L-amygdalin in a traditional Chinese medicine, amygdalin. Background Art

[0002] Amygdalin, whose chemical name is phenylhydroxyacetyl cyanamide-β-D-glucosyl-6-β-D-glucoside, has a structure as shown below.

[0003]

[0004] Amygdalin is mainly found in the fruits and seeds of Rosaceae plants such as apricot, mountain peach, and loquat. It has antitussive and antiasthmatic, anti-ulcer, and anti-tumor effects. There is a chiral carbon atom in its structure, so amygdalin has two diastereomers, namely D-amygdalin and L-amygdalin, of which D-amygdalin is the natural form. The configuration of diastereomers is easily affected by external factors such as acid, alkali, and heating, and diastereomerization occurs; for D-amygdalin, it is easily affected by alkaline conditions and converted into L-amygdalin. At present, research mainly believes that D-amygdalin is the active ingredient, L-amygdalin has no antitumor activity, and other activities have not yet been clarified. There are also obvious differences in the in vitro β-glucosidase hydrolysis of the two.

[0005] Bitter almonds and peach kernels are commonly used Chinese medicines in clinical practice. Both of them contain amygdalin. The 2020 edition of the Chinese Pharmacopoeia uses amygdalin as the content determination indicator. Since a series of complex processing and preparation processes are required from medicinal materials to decoction pieces and then to preparations, D-amygdalin is easily isomerized into L-amygdalin, which affects the efficacy of decoction pieces, granules or prescription preparations. Therefore, separation and analysis of the two isomers can more effectively control the quality of decoction pieces and preparations. However, the current pharmacopoeia has not yet distinguished and stipulated the content standards of D- and L-amygdalin.

[0006] Existing literature reports mostly use C18 or phenyl columns to analyze D / L-amygdalin by reversed-phase liquid chromatography. The method often uses a lower column temperature (such as 20°C) combined with a weaker elution of a low proportion of organic phase (such as 8% acetonitrile). The peak elution time of the target peak is generally more than 20 minutes, so that the separation degree of the target peak pair can reach 1.5; even if a 1.8μm ultra-high performance liquid chromatography column is used for analysis, a relatively satisfactory separation effect cannot be achieved. Therefore, it is challenging to establish a reversed-phase separation method for D- / L-amygdalin, and it plays an important role in practical application and production. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides a liquid chromatography method for rapidly determining D / L-amygdalin in bitter almonds / peach kernels of traditional Chinese medicines. The method has good separation selectivity for the two diastereomers of D / L-amygdalin, fast peak elution of the target peak, and excellent universality, and can be suitable for the determination of D / L-amygdalin in bitter almonds and peach kernels of traditional Chinese medicines at the same time.

[0008] To achieve the above-mentioned purpose, the technical scheme of the present invention is as follows: a liquid chromatography method for rapidly determining D / L-amygdalin in the traditional Chinese medicine bitter almond peach kernel, wherein the chromatographic column stationary phase used in the liquid chromatography method is a biphenyl reverse phase chromatographic filler bonded to a silica gel matrix; wherein the types of the silica gel matrix include fully porous silica gel, core-shell surface porous silica gel, and hybrid silica gel; and the bonded chemical functional group is a biphenyl group.

[0009] Wherein, the biphenyl reverse phase chromatography filler bonded to the silica gel matrix is ​​ChromCore Biphenyl.

[0010] The particle size of the biphenyl reverse phase chromatographic filler bonded to the silica matrix is ​​in the range of 1-50 microns, and the pore size is in the range of 6-100 nanometers. Preferably, the particle size of the biphenyl reverse phase chromatographic filler bonded to the silica matrix is ​​in the range of 1.5-5 microns, and the pore size is in the range of 10-20 nanometers.

[0011] The shape of the silica gel matrix can be spherical or irregular.

[0012] The specifications of the chromatographic column are: column length range 20-600 mm, column inner diameter range 1-1000 mm. Preferably, the specifications of the chromatographic column are: column length range 50-300 mm, column inner diameter range 2.1-4.6 mm.

[0013] Wherein, the parameters of the liquid chromatography method are:

[0014] Column temperature range 20℃-45℃;

[0015] The organic phase is methanol or acetonitrile, and the aqueous phase is acidic aqueous solution or pure water;

[0016] Flow rate range 0.8-3.0mm / s;

[0017] The elution method is isocratic, and an organic phase column flushing process can also be added after the effective isocratic separation stage.

[0018] The aqueous phase is composed of an aqueous solution containing 0.1% phosphoric acid (v / v) or pure water. That is, the mobile phase can be an 8% acetonitrile-water solution or a 20% methanol-water solution, or an 8% acetonitrile-water solution containing 0.1% phosphoric acid or a 20% methanol-water solution containing 0.1% phosphoric acid.

[0019] Among them, the proportion of organic phase is adjusted within the absolute value allowed by the pharmacopoeia ± 2%;

[0020] The elution method is isocratic, and a high proportion of organic phase (95%) can be added to the chromatographic column for flushing after the effective isocratic separation stage to reduce the influence of strongly retained components on subsequent separation.

[0021] The liquid chromatography method adopts a ChromCore Biphenyl 4.6×250mm×5μm or 4.6×150mm×3μm or 2.1×100mm×1.8μm or 2.1×50mm×1.8μm chromatographic column, the mobile phase can be an 8% acetonitrile-water solution or a 20% methanol-water solution, or an 8% acetonitrile-water solution containing 0.1% phosphoric acid or a 20% methanol-water solution containing 0.1% phosphoric acid, the column temperature is 25°C-45°C, and the isocratic time is 3min-30min.

[0022] The liquid chromatography method adopts a ChromCore Biphenyl 4.6×150mm×3μm or 2.1×100mm×1.8μm or 2.1×50mm×1.8μm chromatographic column, the mobile phase can be an 8% acetonitrile-water solution or a 20% methanol-water solution, or an 8% acetonitrile-water solution containing 0.1% phosphoric acid or a 20% methanol-water solution containing 0.1% phosphoric acid, the column temperature is 25°C-45°C, and the isocratic time is 3min-20min.

[0023] The liquid chromatography method uses a ChromCore Biphenyl 2.1×100mm×1.8μm or 2.1×50mm×1.8μm chromatographic column, the mobile phase uses 8% acetonitrile-aqueous solution containing 0.1% phosphoric acid or 20% methanol-aqueous solution containing 0.1% phosphoric acid, the column temperature is 25°C-45°C, and the isocratic time is 3min-6min.

[0024] The liquid chromatography method uses a ChromCore Biphenyl 2.1×50mm×1.8μm chromatographic column, the mobile phase uses 8% acetonitrile-0.1% phosphoric acid aqueous solution or 20% methanol-0.1% phosphoric acid aqueous solution, the column temperature is 25°C-45°C, and the isocratic time is 3min-4min.

[0025] The separation degree of the two diastereomers of D / L-amygdalin by the liquid chromatography method is between 1.8 and 4.22.

[0026] Preferably, the liquid chromatography method uses ChromCore Biphenyl (5μm, 4.6×250mm) with a mobile phase of 8% acetonitrile-water (containing 0.1% phosphoric acid, v / v) solution, one of the optimal column temperatures is 30°C, and the isocratic time is 30min; if the mobile phase is 20% methanol-water (containing 0.1% phosphoric acid, v / v) solution, one of the optimal column temperatures is 45°C, and the isocratic time is 25min.

[0027] Preferably, the liquid chromatography method uses ChromCore Biphenyl (3μm, 4.6×150mm) with a mobile phase of 8% acetonitrile-water (containing 0.1% phosphoric acid, v / v) solution, one of the optimal column temperatures is 28°C, and the isocratic time is 19min; if the mobile phase is 20% methanol-water (containing 0.1% phosphoric acid, v / v) solution, one of the optimal column temperatures is 45°C, and the isocratic time is 16min.

[0028] Preferably, the liquid chromatography method uses ChromCore Biphenyl (1.8 μm, 2.1×100 mm) with an optimal column temperature of 45° C., a mobile phase of 8% acetonitrile-water (containing 0.1% phosphoric acid, v / v) solution or 20% methanol-water (containing 0.1% phosphoric acid, v / v) solution, and an isocratic time of 6 min.

[0029] Preferably, the liquid chromatography method uses ChromCore Biphenyl (1.8 μm, 2.1×50 mm) with a mobile phase of 8% acetonitrile-water (containing 0.1% phosphoric acid, v / v) solution, one of the optimal column temperatures is 25° C., and the isocratic time is 4 min; if the mobile phase is 20% methanol-water (containing 0.1% phosphoric acid, v / v) solution, one of the optimal column temperatures is 45° C., and the isocratic time is 3 min.

[0030] The chromatographic column stationary phase used in the present invention is a biphenyl reverse phase chromatographic filler (such as ChromCore Biphenyl) bonded to a silica matrix. Compared with the C18 used in the current pharmacopoeia and the phenyl reverse phase column in the reference literature, it has superior separation selectivity and can achieve better separation of the two diastereomers of D / L-amygdalin.

[0031] In the present invention, the specifications of the chromatographic column used are: column length ranges from 20 to 600 mm, preferably 50 to 300 mm, column inner diameter ranges from 1 to 1000 mm, preferably 2.1 to 4.6 mm. The chromatographic column tube material can be stainless steel, PEEK or glass.

[0032] In order to achieve baseline separation of D / L-amygdalin in the sample within a shorter analysis time and avoid the influence of interfering peaks in the matrix on the separation, the chromatographic method can obtain the best separation performance by adjusting factors such as column temperature, organic phase type and ratio, flow rate and elution mode:

[0033] For example, the temperature range of the chromatographic column can be 20°C-45°C;

[0034] The organic phase is methanol or acetonitrile, and the aqueous phase is an acidic aqueous solution (e.g., 0.1% phosphoric acid, v / v) or pure water;

[0035] The proportion of organic phase is adjusted within the absolute value allowed by the pharmacopoeia ±2%;

[0036] Flow rate range 0.8-3.0mm / s;

[0037] The elution method is isocratic, and a high proportion of organic phase (95%) can be added to the column for flushing after the effective isocratic separation stage to reduce the influence of strongly retained components on subsequent separation.

[0038] The liquid chromatography method of the present invention has good separation selectivity for the two diastereomers of D / L-amygdalin, with a separation degree between 1.8 and 4.22, and the separation degree is greatly increased. Moreover, the peak elution time of the target peak is greatly shortened, and the isocratic time can be shortened to 3min-6min by using a 2.1×100mm×1.8μm or 2.1×50mm×1.8μm chromatographic column of ChromCoreBiphenyl.

[0039] The method of the invention is simple, rapid, practical, has good reproducibility and universality, and can be suitable for the determination of D / L-amygdalin in the traditional Chinese medicines bitter almond and peach kernel.

[0040] The method of the present invention can be applied on HPLC to UHPLC.

[0041] The method of the invention adopts biphenyl reverse phase chromatographic filler bonded with silica gel matrix as the stationary phase of the chromatographic column, and combines preferred parameters such as chromatographic column temperature and mobile phase type, flow rate, elution method, etc., to achieve rapid determination of D / L-amygdalin in bitter almonds / peach kernels of traditional Chinese medicines, can achieve baseline separation of D / L-amygdalin in samples within a relatively short analysis time, has good separation selectivity for two diastereomers of D / L-amygdalin, has excellent universality, and can be suitable for the determination of D / L-amygdalin in bitter almonds and peach kernels of traditional Chinese medicines at the same time. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 is the chromatogram of Example 1.

[0043] Figure 2 This is the chromatogram of Example 2.

[0044] Figure 3 This is the chromatogram of Example 3.

[0045] Figure 4is the chromatogram of Example 4A.

[0046] Figure 5 is the chromatogram of Example 4B.

[0047] Figure 6 It is the chromatogram of Example 5. Among them, the detection limit (S / N=3:1) and the quantification limit (S / N=10:1) of the D-amygdalin peak are 0.08 μg / mL and 0.29 μg / mL, respectively.

[0048] Figure 7 This is the chromatogram of Example 6.

[0049] Figure 8 This is the chromatogram of Example 7.

[0050] Fig. 9 It is the chromatogram of Example 8.

[0051] Fig.10 It is the chromatogram of Example 9.

[0052] Fig.11 This is the chromatogram of Example 10.

[0053] Fig.12 This is the chromatogram of Example 11.

[0054] Fig.13 is the chromatogram of Example 11B.

[0055] Fig.14 is a linear relationship graph for Example 11C.

[0056] Fig.15 is the chromatogram of Example 11D.

[0057] Fig.16 It is a linear relationship diagram of Example 12.

[0058] Fig.17 This is the chromatogram of Example 12.

[0059] Fig.18 This is the chromatogram of Example 13.

[0060] Fig.19 This is the chromatogram of Example 14.

[0061] Fig. 20 It is the chromatogram of Comparative Example 1.

[0062] Fig.21 It is the chromatogram of Comparative Example 2.

[0063] Fig. 22 It is the chromatogram of Comparative Example 3. DETAILED DESCRIPTION

[0064] The technical solution of the present invention is further described below in conjunction with specific embodiments, but the present invention is not limited to these embodiments. Those skilled in the art should understand that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0065] Sample preparation:

[0066] 1. Amygdalin reference solution: Take an appropriate amount of amygdalin reference, dissolve it in methanol and dilute it to make a reference solution containing 40 μg of amygdalin per 1 mL.

[0067] 2. Conversion solution of L-amygdalin diastereomer: Take 500 μL of amygdalin reference substance (D-amygdalin) solution, place it in a 1.5 mL injection bottle, add 500 μL of sodium hydroxide solution with pH = 11, shake well, let it stand for 0.5 h, and then adjust the pH to neutral with 1 mol / L hydrochloric acid.

[0068] 3. Extract of boiled bitter almond sample: Take 0.3 g of boiled bitter almond sample powder, accurately weigh it, put it in a 50 mL centrifuge tube, accurately add 25 mL of methanol, seal it, weigh it, and treat it ultrasonically (power 250 W, frequency 50 kHz) for 30 minutes. Let it cool, weigh it again, make up the lost weight with methanol, shake it well, filter it, accurately measure 2 mL of the filtrate, put it in a 10 mL volumetric flask, add 50% methanol (v / v) to dilute it to the scale, shake it well, filter it, and take the filtrate.

[0069] 4. Simmered peach kernel sample extract: Take 0.3 g of the steamed peach kernel sample powder, accurately weigh it, put it in a 50 mL centrifuge tube, accurately add 50 mL of 70% methanol (v / v), seal it, weigh it, and treat it with ultrasound (power 250 W, frequency 50 kHz) for 30 minutes. Let it cool, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0070] Example 1. Analysis of D / L-amygdalin in bitter almonds

[0071] Chromatographic column: ChromCore Biphenyl (4.6×250mm, 5μm);

[0072] Mobile phase: Phase A, 0.1% phosphoric acid water (v / v); Phase B, acetonitrile; Isocratic elution: 0-30 min, 8% B;

[0073] Flow rate: 1.0 mL / min;

[0074] Column temperature: 25°C;

[0075] Injection volume: 5 μL;

[0076] Detector: UV 207nm;

[0077] Sample: Bitter almond extract;

[0078] Peak names: 1, L-amygdalin; 2, D-amygdalin.

[0079] The analysis results of Example 1 are shown in Table 1 and Figure 1 The results showed that when the chromatographic column (250×4.6 mm×5 μm) specified in the present invention was used to analyze the bitter almond sample, the separation degree of D / L-amygdalin in the sample was 3.32, and accurate quantitative analysis of the target peak could be achieved.

[0080] Table 1 Typical test results of bitter almond samples

[0081] (Chromatographic column 250×4.6mm×5μm; column temperature 25℃)

[0082]

[0083] Example 2 Analysis of D / L-Amygdalin in Simmered Bitter Almonds

[0084] Chromatographic column: ChromCore Biphenyl (4.6×250mm, 5μm);

[0085] Mobile phase: Phase A, 0.1% phosphoric acid water (v / v); Phase B, acetonitrile; Isocratic elution: 0-30 min, 8% B;

[0086] Flow rate: 1.0 mL / min;

[0087] Column temperature: 30°C

[0088] Injection volume: 5 μL;

[0089] Detector: UV 207nm;

[0090] Sample: boiled bitter almond extract;

[0091] Peak name: 1. L-amygdalin; 2. D-amygdalin

[0092] The analysis results of Example 2 are shown in Table 2 and Figure 2 The chromatographic column (250×4.6 mm×5 μm) specified in the present invention was used to analyze the blanched bitter almond sample, and the difference from Example 1 was the different column temperature. The results showed that the separation degree of D / L-amygdalin in the sample in this example was 3.18, and the column temperature of 30°C was more conducive to the separation of D-amygdalin and the interference peak in the sample, and the accurate quantitative analysis of the target peak could be achieved.

[0093] Table 2 Typical test results of stewed bitter almond samples

[0094] (Chromatographic column 250×4.6mm×5μm; column temperature 30℃)

[0095]

[0096] Example 3 Analysis of D / L-amygdalin in stewed peach kernels

[0097] Chromatographic column: ChromCore Biphenyl (4.6×250mm, 5μm);

[0098] Mobile phase: Phase A, 0.1% phosphoric acid water (v / v); Phase B, acetonitrile; Isocratic elution: 0-30min, 8% B; (Note: To prevent the strong retention components in the sample from affecting the subsequent separation, a column cleaning process was added, 30-30.5min, 8%-95% B; 30.5-35min, 95% B)

[0099] Flow rate: 1.0 mL / min;

[0100] Column temperature: 30°C;

[0101] Injection volume: 5 μL;

[0102] Detector: UV 207nm;

[0103] Sample: Simmered peach kernel extract;

[0104] Peak names: 1, L-amygdalin; 2, D-amygdalin.

[0105] The analysis results of Example 3 are shown in Table 3 and Figure 3 The conventional chromatographic column (250×4.6mm×5μm) specified in the present invention was used to analyze the stewed peach kernel sample. The results showed that the separation degree of D / L-amygdalin in the sample was 3.15. As the column temperature increased, the separation degree of the two decreased slightly. When the column temperature was 30°C, it was more conducive to the separation of D-amygdalin and the interference peak in the sample. The accurate quantitative analysis of D-amygdalin was achieved by fine-tuning the column temperature.

[0106] Table 3 Typical test results of stewed peach kernel samples

[0107] (Chromatographic column 250×4.6mm×5μm; column temperature 30℃)

[0108]

[0109] Example 4A. Rapid method transfer analysis of stewed bitter almonds using chromatographic columns of different particle sizes and specifications

[0110] Chromatographic column: ChromCore Biphenyl (4.6×150mm, 3μm);

[0111] Mobile phase: Phase A, 0.1% phosphoric acid water (v / v); Phase B, acetonitrile; Isocratic elution: 0-19 min, 8% B;

[0112] Flow rate: 1.0 mL / min;

[0113] Column temperature: 28°C;

[0114] Injection volume: 3 μL;

[0115] Detector: UV 207nm;

[0116] Sample: a.Braised bitter almond extract

[0117] b. Amygdalin reference solution

[0118] cL-Amygdalin epimer conversion solution

[0119] Peak names: 1, L-amygdalin; 2, D-amygdalin.

[0120] Example 4A Rapid method conversion analysis of stewed bitter almonds was performed using chromatographic columns of different particle sizes and specifications. The analysis results are shown in Tables 4 and Figure 4 . Under the premise of maintaining column equivalence, chromatographic columns with different particle sizes and specifications were used for rapid method conversion. The results showed that the resolution of D / L-amygdalin in the blanched bitter almond sample was 3.44 when the 150×4.6mm×3μm chromatographic column was used to analyze the sample; the column temperature of 28℃ could improve the separation of D-amygdalin and interference peaks in the sample, and achieve accurate quantitative analysis of the target peak in the sample; the chromatographic peak 1 in the sample was further confirmed by the L-amygdalin diastereomer conversion solution; the analysis time of the converted method was shortened by 11min compared with the conventional method of 250×4.6mm×5μm, which improved the analysis efficiency of the method.

[0121] Table 4. Typical test results of stewed bitter almonds

[0122] (Chromatographic column 4.6×150mm×3μm; column temperature 28℃)

[0123]

[0124] Example 4B. Rapid method conversion analysis of steamed peach kernels using chromatographic columns of different particle sizes and specifications

[0125] Column: ChromCore Biphenyl (4.6×150 mm, 3 μm);

[0126] Mobile phase: Phase A, 0.1% phosphoric acid water (v / v); Phase B, acetonitrile; Isocratic elution: 0-19 min, 8% B;

[0127] Flow rate: 1.0 mL / min;

[0128] Column temperature: 28°C;

[0129] Injection volume: 3 μL;

[0130] Detector: UV 207nm;

[0131] Sample: a.Steamed peach kernel extract

[0132] b. Amygdalin reference solution

[0133] Peak names: 1, L-amygdalin; 2, D-amygdalin.

[0134] Example 4B Rapid method conversion analysis of steamed peach kernels was performed using chromatographic columns of different particle sizes and specifications. The analysis results are shown in Tables 5 and Figure 5 . Under the premise of maintaining column equivalence, chromatographic columns with different particle sizes and specifications were used for rapid method conversion. The results showed that the resolution of D / L-amygdalin in the sample was 3.46 when the 150×4.6mm×3μm chromatographic column was used to analyze the peach kernel sample. The column temperature of 28℃ improved the separation of D-amygdalin and interference peaks in the sample, and the accurate quantitative analysis of the target peak could be achieved. The analysis time of the converted method was shortened by 11min compared with the conventional method of 250×4.6mm×5μm, which improved the analysis efficiency of the method.

[0135] Table 5 Typical test results of stewed peach kernels

[0136] (Chromatographic column 150×4.6mm×3μm; column temperature 28℃)

[0137]

[0138] Example 5. Rapid method conversion analysis of stewed peach kernels and stewed bitter almond samples using chromatographic columns of different particle sizes and specifications

[0139] Column: ChromCore Biphenyl (2.1×100 mm, 1.8 μm);

[0140] Mobile phase: Phase A, 0.1% phosphoric acid water (v / v); Phase B, acetonitrile; Isocratic elution: 0-6min, 8% B;

[0141] Flow rate: 0.6 mL / min;

[0142] Column temperature: 45°C;

[0143] Injection volume: 1 μL;

[0144] Detector: UV 207nm;

[0145] Sample: amygdalin reference solution

[0146] bL-Amygdalin epimer conversion solution

[0147] cSimmered peach kernel extract

[0148] dBraised bitter almond extract

[0149] Peak names: 1, L-amygdalin; 2, D-amygdalin.

[0150] Example 5 Rapid method conversion analysis was performed on the stewed peach kernel and stewed bitter almond samples using chromatographic columns of different particle sizes and specifications. The analysis results are shown in Tables 6 and Figure 6 While maintaining column equivalence, chromatographic columns of different particle sizes and specifications were used for rapid method conversion. The results showed that the separation of D / L-amygdalin in the samples of stewed peach kernels and stewed bitter almonds was greater than 2.5 when the 100×2.1mm×1.8μm chromatographic column was used to analyze the samples. The column temperature of 45℃ improved the separation of D-amygdalin and interference peaks in the samples, and the accurate quantitative analysis of the target peaks could be achieved. The analysis time was shortened by 24min compared with the 250×4.6mm×5μm method, which significantly improved the analysis efficiency of the method.

[0151] Table 6 Test results of stewed peach kernels and stewed bitter almonds

[0152] (Chromatographic column 100×2.1mm×1.8μm; column temperature 45℃)

[0153]

[0154]

[0155] Example 6. Study on the effect of different column temperatures on the separation of D / L-amygdalin

[0156] Column: ChromCore Biphenyl (2.1×100 mm, 1.8 μm);

[0157] Mobile phase: Phase A, 0.1% phosphoric acid water (v / v); Phase B, acetonitrile; Isocratic elution: 8% B;

[0158] Flow rate: 0.6 mL / min;

[0159] Column temperature: 25℃ / 30℃ / 35℃ / 40℃ / 45℃;

[0160] Injection volume: 1 μL;

[0161] Detector: UV 207nm;

[0162] Sample: L-amygdalin diastereomer conversion solution;

[0163] Peak name: 1. L-amygdalin; 2. D-amygdalin

[0164] Example 6 The effect of different column temperatures on the separation of D / L-amygdalin was studied. The analysis results are shown in Tables 7 and Figure 7 The study of the effect of different column temperatures on the separation of D / L-amygdalin showed that temperature had a significant effect on the retention time and resolution of D / L-amygdalin: the higher the temperature, the shorter the retention time and the lower the resolution, but baseline separation can be achieved within the range of 25°C-45°C.

[0165] Table 7. Effect of column temperature on the separation of D / L-amygdalin

[0166]

[0167]

[0168] Example 7 Continuous Sampling Precision Study

[0169] Under the determined optimized conditions, the L-amygdalin diastereomer conversion solution sample was taken and injected 5 times continuously. The results are shown in Table 8 and Figure 8 The results showed that the RSD of the D / L-amygdalin peak retention time was 0.3%; the RSD of the peak area was 0.41% and 0.49%, respectively, indicating that the method of the present invention has good precision.

[0170] Table 8 Continuous injection precision investigation

[0171]

[0172] Example 8. Rapid method conversion using chromatographic columns of different particle sizes and specifications Chromatographic columns: ChromCoreBiphenyl (2.1×50 mm, 1.8 μm);

[0173] Mobile phase: phase A, 0.1% phosphoric acid water (v / v); phase B, acetonitrile; isocratic elution: 0-4min, 8% B;

[0174] Flow rate: 0.6 mL / min;

[0175] Column temperature: 25°C;

[0176] Injection volume: 1 μL;

[0177] Detector: UV 207nm;

[0178] Sample: Amygdalin reference solution

[0179] Bitter almond extract

[0180] L-Amygdalin epimer conversion solution

[0181] Peak names: 1, L-amygdalin; 2, D-amygdalin.

[0182] Example 8 A 50×2.1 mm×1.8 μm chromatographic column was used to analyze the bitter almond sample. The analysis results are shown in Tables 9 and Fig. 9 The results showed that the separation degree of D / L-amygdalin in the sample was >2.3, and the analysis time was 4 minutes, which was 26 minutes shorter than the 250×4.6mm×5μm method, significantly improving the analysis efficiency of the method.

[0183] Table 9 Sample test results

[0184] (Chromatographic column 50×2.1mm×1.8μm)

[0185]

[0186] Example 9. Study on the influence of different mobile phase systems on the separation of D / L-amygdalin

[0187] Column: ChromCore Biphenyl, 1.8 μm;

[0188] Column size: 2.1×100mm;

[0189] Mobile phase: 1. 92 / 8v / v0.1% phosphoric acid solution (v / v) / acetonitrile

[0190] 2. 80 / 20v / v0.1% phosphoric acid solution (v / v) / methanol

[0191] Flow rate: 0.6 mL / min;

[0192] Column temperature: 45°C

[0193] Injection volume: 1μL

[0194] Detector: UV 207nm;

[0195] Instrument: Agilent 1290 Q Infinity II;

[0196] Sample: L-Amygdalin diastereomer conversion solution

[0197] Peak names: 1. L-amygdalin; 2. D-amygdalin;

[0198] Example 9 The effects of different mobile phase systems on the separation of D / L-amygdalin were studied. The results are shown in Tables 10 and Fig.10The study showed that the methanol-0.1% (v / v) phosphoric acid system was better than the acetonitrile-0.1% (v / v) phosphoric acid system in the separation of D / L-amygdalin. The former had a resolution of 3.4 and the latter had a resolution of 2.7. However, due to the greater viscosity of the mobile phase, the system pressure of the former was higher than that of the latter (535 bar and 420 bar).

[0199] Table 10 Test results of samples in different flow systems

[0200]

[0201]

[0202] Example 10. Analysis of actual samples using a conventional ChromCore Biphenyl column (250×4.6 mm×5 μm)

[0203] Chromatographic column: ChromCore Biphenyl (4.6×250mm, 5μm);

[0204] Mobile phase: Phase A, 0.1% phosphoric acid water (v / v); Phase B, methanol; Isocratic elution: 0-25min, 20% B;

[0205] Flow rate: 1.0 mL / min;

[0206] Column temperature: 45°C

[0207] Injection volume: 5 μL;

[0208] Detector: UV 210nm

[0209] Sample: a. Amygdalin reference solution

[0210] bL-Amygdalin epimer conversion solution

[0211] c. Amygdalin extract

[0212] d.Braised bitter almond extract

[0213] e.Steamed peach kernel extract

[0214] Peak name: 1. L-amygdalin; 2. D-amygdalin

[0215] Example 10 A conventional ChromCore Biphenyl column (250×4.6 mm×5 μm) was used to analyze the actual sample. The analysis results are shown in Tables 11 and Fig.11 The results showed that the resolution of D / L-amygdalin in the sample was >3.5, and the separation of D-amygdalin and interference peaks in the sample was good, which could achieve accurate quantitative analysis of the target peak.

[0216] Table 11 Typical analysis results of actual samples

[0217] (Chromatographic column 250×4.6mm×5μm; column temperature 45℃)

[0218]

[0219]

[0220] Example 11. Rapid method transfer study using chromatographic columns of different particle sizes and specifications

[0221] Column: ChromCore Biphenyl (4.6×150 mm, 3 μm);

[0222] Mobile phase: Phase A, 0.1% phosphoric acid water (v / v); Phase B, methanol; Isocratic elution: 0-16 min, 20% B;

[0223] Flow rate: 1.0 mL / min;

[0224] Column temperature: 45°C;

[0225] Injection volume: 5 μL;

[0226] Detector: UV 210nm;

[0227] Sample: a. Amygdalin reference solution

[0228] bL-Amygdalin epimer conversion solution

[0229] c. Amygdalin extract

[0230] d.Braised bitter almond extract

[0231] e.Steamed peach kernel extract

[0232] Peak names: 1, L-amygdalin; 2, D-amygdalin.

[0233] Example 11: Under the premise of maintaining column equivalence, chromatographic columns of different particle sizes and specifications were used for rapid method conversion. The actual sample was analyzed using a chromatographic column of 150×4.6mm×3μm. The analysis results are shown in Tables 12 and Fig.12 The results showed that the separation between D / L-amygdalin and interference peaks in the reference and samples was greater than 3.5, achieving accurate quantitative analysis of the target peaks in the samples; the analysis time of the converted method was shortened by 9 minutes compared with the conventional method of 250×4.6mm×5μm, which improved the analysis efficiency of the method.

[0234] Table 12 Actual sample test results

[0235] (Chromatographic column 150×4.6mm×3μm; column temperature 45℃)

[0236]

[0237] Example 11B. Rapid method transfer using chromatographic columns of different particle sizes and specifications

[0238] Column: ChromCore Biphenyl (2.1×100 mm, 1.8 μm);

[0239] Mobile phase: Phase A, 0.1% phosphoric acid water (v / v); Phase B, methanol; Isocratic elution: 0-6min, 20% B;

[0240] Flow rate: 1.0 mL / min;

[0241] Column temperature: 45°C;

[0242] Injection volume: 1 μL;

[0243] Detector: UV 210nm;

[0244] Sample: aL-Amygdalin epimer conversion solution

[0245] b. Amygdalin extract

[0246] c.Braised bitter almond extract

[0247] d.Steamed peach kernel extract

[0248] e. Amygdalin reference solution

[0249] Peak names: 1, L-amygdalin; 2, D-amygdalin.

[0250] Implementation Case 11B: Under the premise of maintaining column equivalence, chromatographic columns of different particle sizes and specifications were used for rapid method conversion: the actual sample was analyzed using a 100×2.1mm×1.8μm chromatographic column. The analysis results are shown in Tables 13 and Fig.13 The results showed that the separation between D / L-amygdalin and interference peaks in the reference and sample was greater than 3.3, achieving accurate quantitative analysis of the target peaks in the sample; the analysis time of the converted method was shortened by 19 minutes compared with the conventional method of 250×4.6mm×5μm, significantly improving the analysis efficiency of the method.

[0251] Table 13 Sample test results

[0252] (Chromatographic column 100×2.1mm×1.8μm; column temperature 45℃)

[0253]

[0254]

[0255] Example 11C. Investigation of linear relationship and minimum limit of quantification and limit of detection

[0256] The linear range, detection limit and quantification limit were investigated by stepwise dilution of amygdalin reference solution with a concentration of 40 μg / mL. The analysis results are shown in Table 14 and Fig.14 Results The target compound was in the range of 2-40 μg / mL, with a linear correlation coefficient of >0.999, and the limits of quantification and detection were 0.44 μg / mL and 0.13 μg / mL, respectively.

[0257] Table 14

[0258]

[0259] Case Study 11D: Rapid Method Transfer Using Columns of Different Particle Sizes and Formats

[0260] Column: ChromCore Biphenyl (2.1×50 mm, 1.8 μm);

[0261] Mobile phase: Phase A, 0.1% phosphoric acid water (v / v); Phase B, methanol; Isocratic elution: 0-3min, 20% B;

[0262] Flow rate: 1.0 mL / min;

[0263] Column temperature: 45°C;

[0264] Injection volume: 1 μL;

[0265] Detector: UV 210nm;

[0266] Sample: aL-Amygdalin epimer conversion solution

[0267] b. Amygdalin extract

[0268] c.Braised bitter almond extract

[0269] d.Steamed peach kernel extract

[0270] e. Amygdalin reference solution

[0271] Peak names: 1, L-amygdalin; 2, D-amygdalin.

[0272] In order to further improve the analysis efficiency, Example 11D used a 50×2.1 mm×1.8 μm chromatographic column to analyze the actual sample. The analysis results are shown in Tables 15 and Fig.15Results The separation degree of D / L-amygdalin in the reference and sample was greater than 1.8, achieving accurate quantitative analysis of the target peak in the sample; the analysis time of the converted method was shortened by 22 minutes compared with the conventional method of 250×4.6mm×5μm, and the retention time was only less than 2 minutes, which greatly accelerated the analysis speed and significantly improved the analysis efficiency of the method.

[0273] Table 15 Sample test results

[0274] (Chromatographic column 50×2.1mm×1.8μm)

[0275]

[0276]

[0277] Example 12: Using a 150×4.6 mm×3.0 μm chromatographic column and a methanol-water mobile phase system, actual samples were analyzed and the linear range of the target analyte was investigated.

[0278] Column: ChromCore Biphenyl (4.6×150 mm, 3 μm);

[0279] Mobile phase: phase A, water; phase B, methanol; isocratic elution: 0-16 min, 20% B;

[0280] Flow rate: 1.0 mL / min;

[0281] Column temperature: 45°C;

[0282] Injection volume: 5 μL;

[0283] Detector: UV 210nm;

[0284] Sample: a. Amygdalin reference solution

[0285] bL-Amygdalin epimer conversion solution

[0286] c. Amygdalin extract

[0287] d.Steamed peach kernel extract

[0288] Peak names: 1, L-amygdalin; 2, D-amygdalin.

[0289] Example 12 A 150×4.6 mm×3.0 μm chromatographic column was used in combination with a methanol-water mobile phase system to analyze actual samples and examine the linear range of the target analyte. The results are shown in Tables 16 and Fig.16 , 17The results showed that the separation of D / L-amygdalin in the reference and samples was >3.2, and the linear correlation coefficient of D-amygdalin was 0.9999, indicating that the linear correlation of the target was good, and accurate quantitative analysis of the target peak in the sample was achieved.

[0290] Table 16

[0291]

[0292] Example 13. Analyze actual samples using a 150×4.6 mm×3.0 μm chromatographic column combined with an acetonitrile-water mobile phase system

[0293] Column: ChromCore Biphenyl (4.6×150 mm, 3 μm);

[0294] Mobile phase: phase A, water; phase B, acetonitrile; isocratic elution: 0-16 min, 8% B;

[0295] Flow rate: 1.0 mL / min;

[0296] Column temperature: 45°C;

[0297] Injection volume: 5 μL;

[0298] Detector: UV 210nm;

[0299] Sample: a. Amygdalin reference solution

[0300] b. Amygdalin extract

[0301] c.Steamed peach kernel extract

[0302] Peak names: 1, L-amygdalin; 2, D-amygdalin.

[0303] Example 13 A 150×4.6 mm×3.0 μm chromatographic column was used in combination with an acetonitrile-water mobile phase system to analyze actual samples. The analysis results are shown in Tables 17 and Fig.18 The results showed that the resolution of D / L-amygdalin in the reference and samples was >2.5, which fully met the separation requirements and achieved accurate quantitative analysis of the target peaks in the samples.

[0304] Table 17

[0305]

[0306] Example 14. Rapid determination of amygdalin in Huashibaidu granules

[0307] Column: ChromCore Biphenyl (2.1×100 mm, 1.8 μm);

[0308] Mobile phase: Phase A, 0.1% phosphoric acid-water (v / v); Phase B, acetonitrile;

[0309] Gradient Program:

[0310]

[0311] Flow rate: 0.4 mL / min;

[0312] Column temperature: 25°C;

[0313] Injection volume: 1 μL;

[0314] Detector: UV 210nm;

[0315] Sample: a. Amygdalin reference solution

[0316] bL-Amygdalin epimer conversion solution

[0317] c. Boiled amygdalin extract

[0318] d. 70% methanol extract of Huashibaidu granules sample -1

[0319] e. 70% methanol extract of Huashibaidu granules sample -2

[0320] Peak names: 1, L-amygdalin; 2, D-amygdalin.

[0321] Example 14 Rapid determination of amygdalin in Huashibaidu granules was performed: a chromatographic column of 100×2.1 mm×1.8 μm was used in combination with a mobile phase system of acetonitrile-0.1% phosphoric acid water (v / v) to analyze actual samples. The analysis results are shown in Tables 18 and Fig.19 Results The resolution of D / L-amygdalin in the reference substance and the sample was >4.1, and the target peaks in the sample were well separated, achieving accurate quantitative analysis of the target in the sample.

[0322] Table 18 Rapid determination results of amygdalin in Huashibaidu granules

[0323]

[0324]

[0325] Comparative Example 1. Separation and testing of amygdalin in bitter almonds using the method of the 2020 edition of the Chinese Pharmacopoeia Part I

[0326] The chromatographic conditions under the content determination item of bitter almond in the 2020 edition of the Chinese Pharmacopoeia are as follows:

[0327] Chromatographic column: ChromCoreC18 (4.6×250 mm, 5 μm);

[0328] Mobile phase: phase A, 0.1% phosphoric acid water (v / v); phase B, acetonitrile; elution program: 0-30min, 8% B;

[0329] (Note: In order to prevent the strong retention components in the sample from affecting the subsequent separation, a column cleaning process was added: 30-30.5min, 8%-95%B; 30.5-35min, 95%B; 35-35.5min, 95%-8%B; 35.5-40min, 8%B)

[0330] Flow rate: 1.0 mL / min;

[0331] Column temperature: 25°C;

[0332] Injection volume: 10 μL;

[0333] Detection wavelength: UV 207nm;

[0334] Peak names: 1L-amygdalin; 2D-amygdalin.

[0335] Comparative Example 1 Referring to the content determination method under the bitter almond item of the 2020 edition of the Chinese Pharmacopoeia, a C18 chromatographic column was used to respectively simmer the bitter almond sample extraction solution and the conversion solution of the L-amygdalin diastereomer. The analysis results are shown in Tables 19 and Fig. 20 The results showed that the resolution of D / L-amygdalin was only 1.5.

[0336] Table 19. Typical test results of ChromCoreC18 under pharmacopoeia conditions

[0337]

[0338] Comparative Example 2. Separation of amygdalin from bitter almonds using the chromatographic method of the reference

[0339] The chromatographic method of the reference was used, and the chromatographic conditions were as follows:

[0340] Chromatographic column: ChromCore Phenyl (4.6×250 mm, 5 μm);

[0341] Mobile phase: Phase A, 0.1% phosphoric acid water (v / v); Phase B, acetonitrile; Isocratic elution: 0-30 min, 8% B;

[0342] Flow rate: 1.0 mL / min;

[0343] Column temperature: 30°C;

[0344] Injection volume: 10 μL;

[0345] Detection wavelength: UV 210nm;

[0346] Peak names: 1L-amygdalin; 2D-amygdalin.

[0347] References

[0348] 1.Xu Xiuqiong, Liu Jianbo, Wang Shuohui, Zhang Zhiqiang, Liu Xiaohong, Song Jianping, Wang Zhenhua. Determination of the content of two diastereomers of amygdalin by HPLC[J]. Journal of New Chinese Medicines and Clinical Pharmacology, 2019, 30(1):94-98

[0349] 2. Xu Dong, Xu Xiuqiong, Wang Shuohui, Zhang Zhiqiang, Wang Zhenhua, Song Jianping. Study on the stability of two diastereomers of amygdalin[J]. Journal of New Chinese Medicines and Clinical Pharmacology, 2019, 31(3): 359-362.

[0350] Comparative Example 2 According to the chromatographic conditions in the above reference, a phenyl chromatographic column was used to inject bitter almond reference substance, boiled bitter almond sample extraction solution, and L-amygdalin diastereomer conversion solution, respectively. The analysis results are shown in Tables 20 and Fig.21 The results showed that the resolution of D / L-amygdalin could only reach 1.7.

[0351] Table 20. Typical test results for phenyl columns

[0352]

[0353] Note: Since the "strength" of the solvent in the control solution and the sample solution is higher than that of the mobile phase, in order to avoid the influence of the solvent effect, the experiment adopts the "sandwich" injection method to reduce the influence of the solvent effect on the separation.

[0354] Comparative Example 3: Using a phenyl column and a methanol-water mobile phase system to test the separation of D / L-amygdalin

[0355] Chromatographic column: ChromCore Phenyl (4.6×250 mm, 5 μm);

[0356] Mobile phase: phase A, water; phase B, methanol; isocratic elution: 0-25min, 20% B;

[0357] Flow rate: 1.0 mL / min;

[0358] Column temperature: 30°C

[0359] Injection volume: 5 μL;

[0360] Detector: UV 210nm;

[0361] Sample: Conversion solution of L-amygdalin diastereomers;

[0362] Peak names: 1, L-amygdalin; 2, D-amygdalin.

[0363] Comparative Example 3: A phenyl column was used in combination with a methanol-water mobile phase system to feed the conversion solution of the diastereomer of L-amygdalin. The analysis results are shown in Tables 21 and Fig. 22 . Results The separation degree of D / L-amygdalin could only reach 1.06.

[0364] Table 21.

[0365]

[0366] Note: Since the "strength" of the solvent in the control solution and the sample solution is higher than that of the mobile phase, in order to avoid the influence of the solvent effect, the experiment adopts the "sandwich" injection method to reduce the influence of the solvent effect on the separation.

[0367] It can be seen that the chromatographic column stationary phase (such as ChromCore Biphenyl) of the biphenyl reverse phase chromatographic filler bonded to the silica matrix used in the present invention has superior separation selectivity compared with the C18 used in the current pharmacopoeia and the phenyl reverse phase column in the reference literature. Not only is the separation degree greatly increased, the maximum separation degree can reach 4.22, but also the peak time of the target peak is greatly shortened. By using the 2.1×100mm×1.8μm or 2.1×50mm×1.8μm chromatographic column of ChromCore Biphenyl, the isocratic time can be shortened to 3min-6min.

[0368] The method of the invention adopts biphenyl reverse phase chromatographic filler bonded with silica gel matrix as the stationary phase of the chromatographic column, and combines preferred parameters such as chromatographic column temperature and mobile phase type, flow rate, elution method, etc., to achieve rapid determination of D / L-amygdalin in bitter almonds / peach kernels of traditional Chinese medicines, can achieve baseline separation of D / L-amygdalin in samples within a relatively short analysis time, has good separation selectivity for two diastereomers of D / L-amygdalin, has excellent universality, and can be suitable for the determination of D / L-amygdalin in bitter almonds and peach kernels of traditional Chinese medicines at the same time.

[0369] The above-described embodiments are only some preferred solutions of the present invention, but they are not intended to limit the present invention. A person skilled in the relevant technical field may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, any technical solution obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present invention.

Claims

1. A liquid chromatography method for rapid determination of D / L-amygdalin in the traditional Chinese medicine bitter almond peach kernel, characterized in that: The chromatographic column stationary phase used in the liquid chromatography method is a biphenyl reverse phase chromatographic filler bonded to a silica gel matrix; wherein the silica gel matrix types include fully porous silica gel, core-shell surface porous silica gel, and hybrid silica gel; and the bonded chemical functional group is a biphenyl group.

2. The liquid chromatography method for rapidly determining D / L-amygdalin in the Chinese medicine bitter almond peach kernel as claimed in claim 1, characterized in that: The biphenyl reverse phase chromatography filler bonded to the silica gel matrix is ​​ChromCore Biphenyl.

3. The liquid chromatography method for rapidly determining D / L-amygdalin in the Chinese medicine bitter almond peach kernel as claimed in claim 1, characterized in that: The particle size of the biphenyl reverse phase chromatography filler bonded to the silica gel matrix is ​​in the range of 1-50 microns, and the pore size is in the range of 6-100 nanometers.

4. The liquid chromatography method for rapidly determining D / L-amygdalin in the Chinese medicine bitter almond peach kernel as claimed in claim 1, characterized in that: The specifications of the chromatographic column are: column length ranges from 20 to 600 mm, and column inner diameter ranges from 1 to 1000 mm.

5. The liquid chromatography method for rapidly determining D / L-amygdalin in the Chinese medicine bitter almond peach kernel as claimed in claim 1, characterized in that: The parameters of the liquid chromatography method are: Column temperature range 20℃-45℃; The organic phase is methanol or acetonitrile, and the aqueous phase is acidic aqueous solution or pure water; Flow rate range 0.8-3.0mm / s; The elution method is isocratic, and an organic phase column flushing process can also be added after the effective isocratic separation stage.

6. The liquid chromatography method for rapidly determining D / L-amygdalin in the Chinese medicine bitter almond peach kernel as claimed in claim 1, characterized in that: The liquid chromatography method adopts a 4.6×250mm×5μm or 4.6×150mm×3μm or 2.1×100mm×1.8μm or 2.1×50mm×1.8μm chromatographic column of ChromCore Biphenyl, the mobile phase can be an 8% acetonitrile-water solution or a 20% methanol-water solution, or an 8% acetonitrile-water solution containing 0.1% phosphoric acid or a 20% methanol-water solution containing 0.1% phosphoric acid, the column temperature is 25°C-45°C, and the isocratic time is 3min-30min.

7. The liquid chromatography method for rapidly determining D / L-amygdalin in the Chinese medicine bitter almond peach kernel as claimed in claim 6, characterized in that: The liquid chromatography method adopts a 4.6×150mm×3μm or 2.1×100mm×1.8μm or 2.1×50mm×1.8μm chromatographic column of ChromCore Biphenyl, the mobile phase can be an 8% acetonitrile-water solution or a 20% methanol-water solution, or an 8% acetonitrile-water solution containing 0.1% phosphoric acid or a 20% methanol-water solution containing 0.1% phosphoric acid, the column temperature is 25°C-45°C, and the isocratic time is 3min-20min.

8. The liquid chromatography method for rapidly determining D / L-amygdalin in the Chinese medicine bitter almond peach kernel as claimed in claim 6, characterized in that: The liquid chromatography method uses a ChromCore Biphenyl 2.1×100mm×1.8μm or 2.1×50mm×1.8μm chromatographic column, an 8% acetonitrile-water solution containing 0.1% phosphoric acid or a 20% methanol-water solution containing 0.1% phosphoric acid as the mobile phase, a column temperature of 25°C-45°C, and an isocratic time of 3min-6min.

9. The liquid chromatography method for rapidly determining D / L-amygdalin in the Chinese medicine bitter almond peach kernel as claimed in claim 6, characterized in that: The liquid chromatography method uses a 2.1×50mm×1.8μm chromatographic column of ChromCore Biphenyl, an 8% acetonitrile-water solution containing 0.1% phosphoric acid or a 20% methanol-water solution containing 0.1% phosphoric acid as the mobile phase, a column temperature of 25°C-45°C, and an isocratic time of 3min-4min.

10. The liquid chromatography method for rapidly determining D / L-amygdalin in the Chinese medicine bitter almond peach kernel according to any one of claims 1 to 9, characterized in that: The separation degree of the two diastereomers of D / L-amygdalin by the liquid chromatography method is between 1.8 and 4.22.

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