Chinese Journal of Chromatography ›› 2021, Vol. 39 ›› Issue (7): 708-714.DOI: 10.3724/SP.J.1123.2020.12009
• Articles • Previous Articles Next Articles
SHEN Jie1, SHEN Wei1, CAI Xue2, WANG Jingxia1, ZHENG Minxia1,*()
Received:
2020-12-09
Online:
2021-07-08
Published:
2021-06-02
Contact:
ZHENG Minxia
Supported by:
CLC Number:
SHEN Jie, SHEN Wei, CAI Xue, WANG Jingxia, ZHENG Minxia. High performance liquid chromatographic method for determination of active components in lithospermum oil and its application to process optimization of lithospermum oil prepared by supercritical fluid extraction[J]. Chinese Journal of Chromatography, 2021, 39(7): 708-714.
Add to citation manager EndNote|Ris|BibTeX
URL: https://www.chrom-china.com/EN/10.3724/SP.J.1123.2020.12009
Fig. 2 HPLC profiles of lithospermum oil standards and samples under optimized chromatographic conditions 1. shikonin; 2. acetylshikonin; 3. β-acetoxyisovaleryl akanin; 4. isobutyryl shikonin; 5. β,β-dimethylacryl shikonin; 6. 2-methylbutyryl.
Compound | tR/min | Linear range/(μg/L) | Calibration curve | r | LOD/(μg/L) | LOQ/(μg/L) |
---|---|---|---|---|---|---|
Shikonin | 6.08±0.01 | 10.00-50.00 | Y=79583X-90506 | 0.9990 | 1.73 | 5.78 |
Acetylshikonin | 9.76±0.02 | 50.00-250.00 | Y=84361X+139619 | 0.9997 | 0.75 | 4.08 |
β-Acetoxyisovaleryl akanin | 14.45±0.01 | 40.00-200.00 | Y=71421X-81331 | 0.9999 | 2.24 | 6.38 |
Isobutyryl shikonin | 18.03±0.03 | 60.00-300.00 | Y=81910X-7028.4 | 0.9985 | 1.58 | 6.55 |
β,β-Dimethylacryl shikonin | 21.81±0.05 | 50.00-250.00 | Y=109607X-699084 | 0.9990 | 2.63 | 7.67 |
2-Methylbutyryl | 24.28±0.05 | 100.00-500.00 | Y=75658X-935990 | 0.9991 | 4.62 | 20.5 |
Table 1 Linear relationships, limits of detection (LODs), and limits of quantification (LOQs) of the six active components (n=3)
Compound | tR/min | Linear range/(μg/L) | Calibration curve | r | LOD/(μg/L) | LOQ/(μg/L) |
---|---|---|---|---|---|---|
Shikonin | 6.08±0.01 | 10.00-50.00 | Y=79583X-90506 | 0.9990 | 1.73 | 5.78 |
Acetylshikonin | 9.76±0.02 | 50.00-250.00 | Y=84361X+139619 | 0.9997 | 0.75 | 4.08 |
β-Acetoxyisovaleryl akanin | 14.45±0.01 | 40.00-200.00 | Y=71421X-81331 | 0.9999 | 2.24 | 6.38 |
Isobutyryl shikonin | 18.03±0.03 | 60.00-300.00 | Y=81910X-7028.4 | 0.9985 | 1.58 | 6.55 |
β,β-Dimethylacryl shikonin | 21.81±0.05 | 50.00-250.00 | Y=109607X-699084 | 0.9990 | 2.63 | 7.67 |
2-Methylbutyryl | 24.28±0.05 | 100.00-500.00 | Y=75658X-935990 | 0.9991 | 4.62 | 20.5 |
Compound | Intra-day precision | Inter-day precision | |||
---|---|---|---|---|---|
Content/ (μg/L) | RSD/ % | Content/ (μg/L) | RSD/ % | ||
Shikonin | 29.35 | 2.17 | 38.89 | 3.70 | |
Acetylshikonin | 150.29 | 0.19 | 197.06 | 1.47 | |
β-Acetoxyisovaleryl akanin | 120.41 | 0.35 | 160.54 | 3.41 | |
Isobutyryl shikonin | 186.86 | 3.81 | 231.60 | 4.49 | |
β,β-Dimethylacryl shikonin | 148.28 | 1.14 | 193.46 | 3.27 | |
2-Methylbutyryl | 293.96 | 2.01 | 393.40 | 2.65 |
Table 2 Test results of intra-day and inter-day precisions (n=6)
Compound | Intra-day precision | Inter-day precision | |||
---|---|---|---|---|---|
Content/ (μg/L) | RSD/ % | Content/ (μg/L) | RSD/ % | ||
Shikonin | 29.35 | 2.17 | 38.89 | 3.70 | |
Acetylshikonin | 150.29 | 0.19 | 197.06 | 1.47 | |
β-Acetoxyisovaleryl akanin | 120.41 | 0.35 | 160.54 | 3.41 | |
Isobutyryl shikonin | 186.86 | 3.81 | 231.60 | 4.49 | |
β,β-Dimethylacryl shikonin | 148.28 | 1.14 | 193.46 | 3.27 | |
2-Methylbutyryl | 293.96 | 2.01 | 393.40 | 2.65 |
Compound | Repeatability RSD/% | Stability RSD/% |
---|---|---|
Shikonin | 1.92 | 0.36 |
Acetylshikonin | 0.33 | 1.50 |
β-Acetoxyisovaleryl akanin | 0.80 | 1.57 |
Isobutyryl shikonin | 0.55 | 2.16 |
β,β-Dimethylacryl shikonin | 0.52 | 2.49 |
2-Methylbutyryl | 0.33 | 3.26 |
Table 3 Test results of repeatability and stability (n=6)
Compound | Repeatability RSD/% | Stability RSD/% |
---|---|---|
Shikonin | 1.92 | 0.36 |
Acetylshikonin | 0.33 | 1.50 |
β-Acetoxyisovaleryl akanin | 0.80 | 1.57 |
Isobutyryl shikonin | 0.55 | 2.16 |
β,β-Dimethylacryl shikonin | 0.52 | 2.49 |
2-Methylbutyryl | 0.33 | 3.26 |
Compound | Background/ (μg/L) | Added/ (μg/L) | Found/ (μg/L) | Recovery/ % | RSD/ % |
---|---|---|---|---|---|
Shikonin | 10.27 | 8.50 | 18.15 | 92.68 | 4.25 |
10.27 | 10.00 | 20.13 | 98.58 | ||
10.27 | 12.50 | 22.85 | 100.65 | ||
Acetylshikonin | 86.63 | 70.00 | 152.95 | 94.74 | 5.35 |
86.63 | 86.50 | 171.31 | 97.89 | ||
86.63 | 104.00 | 195.93 | 105.09 | ||
β-Acetoxyisovaleryl | 71.42 | 57.50 | 123.43 | 90.45 | 4.19 |
akanin | 71.42 | 71.50 | 141.70 | 98.29 | |
71.42 | 85.50 | 153.08 | 95.50 | ||
Isobutyryl shikonin | 63.14 | 50.50 | 111.68 | 96.12 | 7.16 |
63.14 | 63.00 | 132.08 | 109.42 | ||
63.14 | 75.50 | 145.26 | 108.77 | ||
β,β-Dimethylacryl | 75.81 | 60.50 | 132.57 | 93.81 | 8.35 |
shikonin | 75.81 | 75.50 | 149.23 | 97.24 | |
75.81 | 90.50 | 175.10 | 109.71 | ||
2-Methylbutyryl | 148.17 | 118.50 | 251.29 | 87.02 | 4.76 |
148.17 | 148.00 | 289.20 | 95.29 | ||
148.17 | 178.00 | 314.86 | 93.64 |
Table 4 Recovery test results of the HPLC method (n=6)
Compound | Background/ (μg/L) | Added/ (μg/L) | Found/ (μg/L) | Recovery/ % | RSD/ % |
---|---|---|---|---|---|
Shikonin | 10.27 | 8.50 | 18.15 | 92.68 | 4.25 |
10.27 | 10.00 | 20.13 | 98.58 | ||
10.27 | 12.50 | 22.85 | 100.65 | ||
Acetylshikonin | 86.63 | 70.00 | 152.95 | 94.74 | 5.35 |
86.63 | 86.50 | 171.31 | 97.89 | ||
86.63 | 104.00 | 195.93 | 105.09 | ||
β-Acetoxyisovaleryl | 71.42 | 57.50 | 123.43 | 90.45 | 4.19 |
akanin | 71.42 | 71.50 | 141.70 | 98.29 | |
71.42 | 85.50 | 153.08 | 95.50 | ||
Isobutyryl shikonin | 63.14 | 50.50 | 111.68 | 96.12 | 7.16 |
63.14 | 63.00 | 132.08 | 109.42 | ||
63.14 | 75.50 | 145.26 | 108.77 | ||
β,β-Dimethylacryl | 75.81 | 60.50 | 132.57 | 93.81 | 8.35 |
shikonin | 75.81 | 75.50 | 149.23 | 97.24 | |
75.81 | 90.50 | 175.10 | 109.71 | ||
2-Methylbutyryl | 148.17 | 118.50 | 251.29 | 87.02 | 4.76 |
148.17 | 148.00 | 289.20 | 95.29 | ||
148.17 | 178.00 | 314.86 | 93.64 |
Level | Extraction pressure/MPa | Extraction temperature/℃ | CO2 flow/ (L/h) | Blank control |
---|---|---|---|---|
1 | 18 | 30 | 27 | 1 |
2 | 23 | 35 | 30 | 2 |
3 | 28 | 40 | 32 | 3 |
Table 5 Factor level table of orthogonal test
Level | Extraction pressure/MPa | Extraction temperature/℃ | CO2 flow/ (L/h) | Blank control |
---|---|---|---|---|
1 | 18 | 30 | 27 | 1 |
2 | 23 | 35 | 30 | 2 |
3 | 28 | 40 | 32 | 3 |
Test No. | Extraction pressure | Extraction temperature | CO2 Flow | Blank control |
---|---|---|---|---|
1 | 1(18 MPa) | 1(30 ℃) | 1(27 L/h) | 1 |
2 | 1(18 MPa) | 2(35 ℃) | 2(30 L/h) | 2 |
3 | 1(18 MPa) | 3(40 ℃) | 3(32 L/h) | 3 |
4 | 2(23 MPa) | 1(30 ℃) | 2(30 L/h) | 3 |
5 | 2(23 MPa) | 2(35 ℃) | 3(32 L/h) | 1 |
6 | 2(23 MPa) | 3(40 ℃) | 1(27 L/h) | 2 |
7 | 3(28 MPa) | 1(30 ℃) | 3(32 L/h) | 2 |
8 | 3(28 MPa) | 2(35 ℃) | 1(27 L/h) | 3 |
9 | 3(28 MPa) | 3(40 ℃) | 2(30 L/h) | 1 |
Table 6 Orthogonal test for optimization of the extraction process of lithospermum oil by supercritical fluid extraction
Test No. | Extraction pressure | Extraction temperature | CO2 Flow | Blank control |
---|---|---|---|---|
1 | 1(18 MPa) | 1(30 ℃) | 1(27 L/h) | 1 |
2 | 1(18 MPa) | 2(35 ℃) | 2(30 L/h) | 2 |
3 | 1(18 MPa) | 3(40 ℃) | 3(32 L/h) | 3 |
4 | 2(23 MPa) | 1(30 ℃) | 2(30 L/h) | 3 |
5 | 2(23 MPa) | 2(35 ℃) | 3(32 L/h) | 1 |
6 | 2(23 MPa) | 3(40 ℃) | 1(27 L/h) | 2 |
7 | 3(28 MPa) | 1(30 ℃) | 3(32 L/h) | 2 |
8 | 3(28 MPa) | 2(35 ℃) | 1(27 L/h) | 3 |
9 | 3(28 MPa) | 3(40 ℃) | 2(30 L/h) | 1 |
Test No. | Shikonin | Acetylshikonin | β-Acetoxyisovaleryl akanin |
---|---|---|---|
1 | 10.90±0.29 | 93.85±6.54 | 88.18±5.94 |
2 | 11.71±0.12 | 109.76±4.09 | 93.64±2.18 |
3 | 13.98±0.27 | 155.94±8.15 | 129.43±3.77 |
4 | 13.79±0.26 | 137.60±0.46 | 113.74±0.91 |
5 | 20.54±0.39 | 172.14±3.02 | 144.89±6.02 |
6 | 19.45±0.12 | 170.89±3.61 | 139.91±1.05 |
7 | 15.81±0.15 | 152.83±2.10 | 121.70±0.71 |
8 | 13.01±0.25 | 132.91±1.06 | 106.54±0.64 |
9 | 14.03±0.15 | 150.57±0.71 | 123.75±0.15 |
Test No. | Isobutyryl shikonin | β,β-Dimethylacryl shikonin | 2-Methylbutyryl |
1 | 79.67±4.37 | 105.91±5.73 | 184.08±6.54 |
2 | 85.43±2.20 | 104.63±2.07 | 199.47±4.87 |
3 | 116.07±2.79 | 129.33±2.99 | 273.99±7.01 |
4 | 101.15±0.56 | 137.53±0.72 | 234.61±0.78 |
5 | 125.72±1.21 | 151.22±2.31 | 295.33±4.63 |
6 | 130.06±1.19 | 221.44±2.34 | 296.09±2.33 |
7 | 114.45±0.23 | 213.82±1.56 | 261.93±1.32 |
8 | 94.29±0.62 | 145.16±0.72 | 221.70±1.66 |
9 | 105.39±0.30 | 161.81±0.91 | 253.99±0.58 |
Table 7 Active component contents of lithospermum oil prepared by nine supercritical fluid extraction processes (n=3) μg/L
Test No. | Shikonin | Acetylshikonin | β-Acetoxyisovaleryl akanin |
---|---|---|---|
1 | 10.90±0.29 | 93.85±6.54 | 88.18±5.94 |
2 | 11.71±0.12 | 109.76±4.09 | 93.64±2.18 |
3 | 13.98±0.27 | 155.94±8.15 | 129.43±3.77 |
4 | 13.79±0.26 | 137.60±0.46 | 113.74±0.91 |
5 | 20.54±0.39 | 172.14±3.02 | 144.89±6.02 |
6 | 19.45±0.12 | 170.89±3.61 | 139.91±1.05 |
7 | 15.81±0.15 | 152.83±2.10 | 121.70±0.71 |
8 | 13.01±0.25 | 132.91±1.06 | 106.54±0.64 |
9 | 14.03±0.15 | 150.57±0.71 | 123.75±0.15 |
Test No. | Isobutyryl shikonin | β,β-Dimethylacryl shikonin | 2-Methylbutyryl |
1 | 79.67±4.37 | 105.91±5.73 | 184.08±6.54 |
2 | 85.43±2.20 | 104.63±2.07 | 199.47±4.87 |
3 | 116.07±2.79 | 129.33±2.99 | 273.99±7.01 |
4 | 101.15±0.56 | 137.53±0.72 | 234.61±0.78 |
5 | 125.72±1.21 | 151.22±2.31 | 295.33±4.63 |
6 | 130.06±1.19 | 221.44±2.34 | 296.09±2.33 |
7 | 114.45±0.23 | 213.82±1.56 | 261.93±1.32 |
8 | 94.29±0.62 | 145.16±0.72 | 221.70±1.66 |
9 | 105.39±0.30 | 161.81±0.91 | 253.99±0.58 |
Fig. 3 Chromatograms of lithospermum oil prepared by nine supercritical extraction processes Samples S1-S9: obtained from Tests 1-9 in Table 6. 1. shikonin; 2. acetylshikonin; 3. β-acetoxyisovaleryl akanin; 4. isobutyryl shikonin; 5. β,β-dimethylacryl shikonin; 6. 2-methylbutyryl.
|
Viewed | ||||||
Full text |
|
|||||
Abstract |
|
|||||