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Journal of Peking University(Health Sciences) ›› 2019, Vol. 51 ›› Issue (3): 467-476. doi: 10.19723/j.issn.1671-167X.2019.03.014

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Spectrometric analyses of larotaxel and larotaxel liposomes quantification by high performance liquid chromatography

Xue-qi LI1,Jian-wei LI1,2,3,4,Qiu-hong LI1,4,Yan YAN1,Jia-lun DUAN1,Yi-nuo CUI1,Zhan-bo SU1,Qian LUO1,Jia-rui XU1,Ya-fei DU1,Gui-ling WANG1,Ying XIE1,Wan-liang LU1△()   

  • Received:2019-03-14 Online:2019-06-18 Published:2019-06-26
  • Supported by:
    Supported by the National Key Grant for New Drug Innovation 2018ZX09301-018-004

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Abstract: Objective: Larotaxel is a new chemical structure drug, which has not been marketed worldwide. Accordingly, the standard identification and quantification methods for larotaxel remain unclear. The spectrometric analyses were performed for verifying weight molecular formula, molecular weight and chemical structure of larotaxel. Besides, a quantification method was developed for measuring larotaxel in the liposomes.Methods: The molecular formula, molecular weight and chemical structure of larotaxel were studied by using mass spectrometry (MS), infra-red (IR), nuclear magnetic resonance (NMR) and ultraviolet-visible (UV-vis) spectrometric techniques. The absorption wavelength of larotaxel was investigated by UV-vis spectrophotometry full-wavelength scanning. Besides, a quantification method was developed by high performance liquid chromatography (HPLC), and then validated by measuring the encapsulation efficacy of larotaxel liposomes. Results: The four spectral characteristics of larotaxel were revealed and the corresponding standard spectra were defined. It was confirmed that larotaxel had the structure of tricyclic diterpenoids, with the molecular formula of C45H53NO14, the molecular weight of 831.900 1, and the maximum absorption wavelength of 230 nm. The quantitative method of larotaxel was established by using HPLC with a reversed phase C18 column (5 μm, 250 mm×4.6 mm), a mobile phase of acetonitrile-water (75 ∶25, volume/volume), and a detection wavelength of 230 nm. The validation study exhibited that the established HPLC method was stable, and had a high recovery and precision in the quantitative measurement of larotaxel in liposomes. In addition, a new kind of larotaxel liposomes was also successfully prepared. The particle size of the liposomes was about 105 nm, with an even size distribution. And the encapsulation efficiency of larotaxel in the liposomes was above 80%.Conclusion: The present study offers reference standard spectra of larotaxel, including MS, IR, NMR, and UV-vis, and confirms the molecular formula, molecular weight and chemical structure of larotaxel. Besides, the study develops a rapid HPLC method for quality control of larotaxel liposomes.

Key words: Larotaxel, Spectrometric analysis, High performance liquid chromatography

CLC Number: 

  • R927

Figure 1

Mass spectrums of larotaxel by quadrupole-time of flight mass spectrometry (Q-TOF-MS) A, positive ion mode; B, negative ion mode."

Figure 2

Infra-red absorption spectrum of larotaxel"

Figure 3

13C NMR and 1H NMR spectrums of larotaxel A, 13C NMR spectrum of larotaxel; B, 1H NMR spectrum of larotaxel. NMR, nuclear magnetic resonance; ppm, one millionth chemical shift."

Figure 4

2D-NMR spectrums of larotaxel A,1H-1H chemical correlation NMR (1H-1H COSY) spectrum; B, 1H detected heteronuclear singular quantum correlation (HSQC) NMR spectrum; C, 1H detected heteronuclear multiple bond correlation (HMBC) NMR spectrum; D, distortionless enhanced polarization transfer (DEPT) 135 NMR spectrum. NMR, nuclear magnetic resonance; ppm, one millionth chemical shift."

Figure 5

The structural formula and the relative molecular mass of larotaxel"

Table 1

1H NMR and 13C NMR assignments for larotaxel"

Positon Larotaxel
13C 1H
1 79.40
2 80.06 5.68 (1H, d, J=7.7 Hz)
3 38.56 4.07 (1H, d, J=7.7 Hz)
4 79.60
5 84.86 4.75 (1H, d, J=3.3 Hz)
6 26.06 2.46 (1H, dt, J=16.1, 4.4 Hz)
7 32.04 1.38 (1H, m)
8 35.12
9 201.79
10 75.69 6.35 (1H, s)
11 133.63
12 140.40
13 72.17 6.29 (1H, t, J=8.4 Hz)
14 35.86 2.40 (1H, m) 2.23 (1H, m)
15 42.92
16 26.06 1.28 (3H, s)
17 21.48 1.30 (3H, s)
18 14.59 1.87 (3H, s)
19 15.63 2.25 (1H, m)
1.66 (1H, t, J=5.8 Hz)
20 75.43 4.32 (1H, d, J=8.8 Hz)
4.03 (1H, d, J=8.8 Hz)
1' 172.80
2' 73.74 4.63 (1H,br.s)
3' 56.00 5.32 (1H, d, J=8.8 Hz)
4' 138.50
5', 9' 126.61 7.39 (2H, d, J=7.0 Hz)
6', 8' 128.88 7.35 (2H, t, J=7.0 Hz)
7' 128.04 7.33 (1H, t, J=7.0 Hz)
10' 155.20
11' 80.10
11'-(CH3)3 28.12 1.28 (9H, s)
1'' 167.49
2'' 129.26
3'',7'' 130.30 8.16 (2H, d, J=7.7 Hz)
4'',6'' 128.73 7.51 (2H, t, J=7.7 Hz)
5'' 133.63 7.61 (1H, t, J=7.7 Hz)
4-OAc 22.26, 169.70 2.45 (3H, s)
10-OAc 20.87, 169.64 2.27 (3H, s)
1-OH 2.07 (1H, s)
2'-OH 3.31 (1H,br.s)
3'-NH 5.39 (1H, br.d,
J=9.5 Hz)

Figure 6

Ultra-violet visible spectrophotometric scanning spectrum of larotaxel methanol solution (250 mg/L) in the range of 200-800 nm"

Figure 7

Correlation profile between concentration and peak area values of larotaxel measured by HPLC-UV method and typical chromatographs Column: SB-C18 (5 μm, 250 mm×4.6 mm); mobile phase: acetonitrile-water (75 ∶25, volume/volume); detection wavelength: 230 nm. A, correlation profile between concentration and peak area values of larotaxel measured by HPLC-UV method; B, typical chromatogram of laro-taxel measured by HPLC-UV spectrum; C, chromatogram of limit of quantitation of larotaxel measured by HPLC. UV, ultraviolet; HPLC, high performance liquid chromatography."

Table 2

HPLC-UV measurement stability of larotaxel in methanol"

Concentration/(mg/L) Peak area/(mAU·min)
0 h 2 h 4 h 6 h 8 h RSD/%
5.0 59.1±0.0 59.1±0.1 59.1±0.0 59.1±0.1 59.1±0.1 1.2
10.0 118.2±0.1 118.2±0.1 118.2±0.0 118.2±0.1 118.2±0.2 1.9
25.0 295.5±0.2 295.5±0.1 295.5±0.1 295.5±0.2 295.5±0.1 1.7

Table 3

Recoveries of standard larotaxel and larotaxel in the liposomes measured by HPLC-UV"

Substances Concentration/(mg/L) Recovery/% RSD/%
Larotaxel1
 5.0 99.62±0.39 0.51
10.0 99.17±0.10 0.33
25.0 101.34±0.07 0.16
Larotaxel2
 5.0 98.07±0.93 1.21
10.0 99.61±0.06 0.03
25.0 102.68±0.15 0.14

Table 4

Precisions of standard larotaxel and larotaxel in the liposomes measured by HPLC-UV"

Concentration added/(mg/L) Intra-day (n=5) Inter-day (n=5)
Precision/% RSD/% Precision/% RSD/%
2.01 99.23±0.49 1.26 98.00±1.05 1.37
10.01 99.69±0.22 0.31 98.02±2.76 1.20
50.01 98.89±0.43 0.25 98.32±2.03 0.75
2.02 98.41±1.49 1.43 98.03±2.03 1.73
10.02 99.70±0.83 0.09 98.11±1.76 1.02
50.02 99.64±0.33 0.12 98.23±1.53 1.39

Figure 8

Transmission electron microscope (TEM) image of larotaxel liposomes"

Table 5

Physicochemical characterization of larotaxel liposomes"

Formulations Particle size/nm PDI Zeta potential/mV Encapsulation efficiency/%
Blank liposomes 82.13±1.94 0.131±0.032 0.25±0.01
Larotaxel liposomes 105.73±2.30 0.190±0.072 0.24±0.02 87.73±2.70
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