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High performance liquid chromatography analysis method for separating and determining L-alanine isopropyl ester hydrochloride enantiomer

A technology of isopropyl alanine and high performance liquid chromatography, applied in the field of separation and determination of enantiomers of L-alanine isopropyl hydrochloride, high performance liquid chromatography, can solve the problem of being sensitive to other substances Interference, difficult control of derivatization time, poor solution stability and other problems, to achieve the effect of short analysis time, good peak shape symmetry and high recovery rate

Pending Publication Date: 2022-06-24
YAOPHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

One is to use ion-pair mobile phase (perchloric acid) for elution at a wavelength of 205nm, and a chiral chromatographic column filled with crown ether compounds bonded to silica gel for separation. This method has poor sensitivity and low-band detection. Susceptible to interference from other substances, the scope of application is small
The other is to use o-phthalaldehyde and N-acetyl-L-cysteine ​​as the derivatization reagent to carry out the derivatization reaction and detect after derivatization, and derivatize in a phosphate solution with a pH of 7.0. As the reaction time prolongs, the L - The content of isomeric derivatives of isopropyl alanine hydrochloride increases first and then decreases, and the derivation time is not easy to control; and the solution stability is poor, only 2.5 h

Method used

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  • High performance liquid chromatography analysis method for separating and determining L-alanine isopropyl ester hydrochloride enantiomer
  • High performance liquid chromatography analysis method for separating and determining L-alanine isopropyl ester hydrochloride enantiomer
  • High performance liquid chromatography analysis method for separating and determining L-alanine isopropyl ester hydrochloride enantiomer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Selection of chromatographic conditions

[0035] 1.1 The choice of wavelength

[0036] Mobile phase: n-hexane-absolute ethanol-diethylamine ratio is 80:20:0.1.

[0037] Preparation of D-alanine isopropyl hydrochloride derivative solution: Weigh about 10 mg of D-alanine isopropyl hydrochloride derivative, put it in a 10 ml measuring flask, add mobile phase as a diluent to dissolve, And diluted to the mark, shake well, that is.

[0038] Take the D-alanine isopropyl ester hydrochloride derivative solution as the test sample, use the mobile phase as the blank calibration solution, and scan at 190nm~400nm according to the ultraviolet-visible spectrophotometry (Chinese Pharmacopoeia 2020 edition). The results show that the test solution has a maximum absorption peak at 225nm. Therefore, 225nm±5nm was determined as the detection wavelength.

[0039] 1.2 Durability test of the method

[0040] In this test, the chromatographic method of this test is screened by ad...

Embodiment 2

[0052] Example 2 Stability of the solution

[0053] Chromatographic conditions: Same as scheme 1 of Example 1.

[0054] Preparation of the solution:

[0055] Need testing solution: prepare according to the same method of 100% concentration level need testing solution in embodiment 1;

[0056] Control solution: Precisely pipette 1ml of the test solution, put it in a 100ml measuring bottle, dilute it to the mark with diluent, shake well, then precisely pipette 1ml, put it in a 20ml measuring bottle, dilute to the mark with mobile phase as a diluent, and shake well , filtered with a 0.22 μm organic filter, and the subsequent filtrate was obtained.

[0057] At 0 hours, 4 hours, 8 hours, 12 hours, and 24 hours, 10 μl of the test solution and the control solution were accurately measured and tested, and the chromatograms at different time points were recorded. The results are shown in Table 3.

[0058] Table 3 Solution stability results

[0059]

[0060] The results showed t...

Embodiment 3

[0061] Example 3 Preparation of standard curve

[0062] Chromatographic conditions: Same as scheme 1 of Example 1.

[0063] Preparation of the solution:

[0064] Linear stock solution: take about 20 mg of L-alanine isopropyl ester hydrochloride derivative and D-alanine isopropyl ester hydrochloride derivative reference substance, respectively, put them in 10 ml measuring flasks, and add mobile phase as Dissolve the diluent and dilute it to the mark, shake well; precisely measure 1ml each, put it in the same 100ml measuring bottle, dilute it to the mark with mobile phase, and shake well to get it;

[0065] Standard curve solution: Dilute with mobile phase linear stock solution to prepare solutions of L-alanine isopropyl hydrochloride derivatives and D-alanine isopropyl hydrochloride derivatives at different concentrations, see Table 4 :

[0066] Table 4 Preparation of standard curve solution

[0067]

[0068] The results showed that the L-alanine isopropyl ester hydrochl...

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Abstract

The invention discloses a method for determining the content of an enantiomer of L-alanine isopropyl ester hydrochloride by adopting a high performance liquid chromatography, which comprises the following steps: taking a chiral chromatographic column taking amylose bonded silica gel as a filler, taking n-hexane-absolute ethyl alcohol added with 0.1% of diethylamine as a mobile phase, and controlling the volume ratio of the n-hexane to the absolute ethyl alcohol to be (75: 25)-(85: 15); the detection wavelength is 225 + / -5nm; the flow velocity is 1.0 ml / min-1. 2ml / min, and benzoyl chloride is used as a derivatization reagent to prepare a sample. By adopting the method, the L-alanine isopropyl ester hydrochloride isomer can be effectively separated and accurately quantified. The method has the advantages of strong specificity, good precision and high accuracy, and can rapidly and accurately determine the content of the L-alanine isopropyl ester hydrochloride enantiomer.

Description

Technical field [0001] The present invention relates to a detection method for drugs, and specifically relates to a high-performance liquid chromatography analysis method for separating and determining the enantiomers of L-alanine isopropyl ester hydrochloride. Background technique [0002] As an important starting material, L-alanine isopropyl ester hydrochloride has a chiral center and is widely used in the synthesis of drugs, such as tenofovir alafenamide fumarate (TAF). Enantiomeric impurities of L-alanine isopropyl ester hydrochloride D-alanine isopropyl hydrochloride is one of the main sources of chiral impurities in TAF, and its enantiomers need to be strictly controlled content. [0003] [0004] In recent years, the requirements for isomers in drugs have become more and more stringent, not just the limit requirements, but the accurate determination of their content. Currently, there are no pharmacopoeial standards for L-alanine isopropyl ester hydrochloride and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06G01N30/74
CPCG01N30/02G01N30/06G01N30/74
Inventor 钟家庆郭云程周曌唐铨刘强
Owner YAOPHARMA CO LTD
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