CN103163258B - A kind of method measuring trace Triptorelin - Google Patents
A kind of method measuring trace Triptorelin Download PDFInfo
- Publication number
- CN103163258B CN103163258B CN201110409108.2A CN201110409108A CN103163258B CN 103163258 B CN103163258 B CN 103163258B CN 201110409108 A CN201110409108 A CN 201110409108A CN 103163258 B CN103163258 B CN 103163258B
- Authority
- CN
- China
- Prior art keywords
- triptorelin
- minute
- volume ratio
- acid
- methyl alcohol
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- VXKHXGOKWPXYNA-PGBVPBMZSA-N triptorelin Chemical compound C([C@@H](C(=O)N[C@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N1[C@@H](CCC1)C(=O)NCC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@H]1NC(=O)CC1)C1=CC=C(O)C=C1 VXKHXGOKWPXYNA-PGBVPBMZSA-N 0.000 title claims abstract description 55
- 108010050144 Triptorelin Pamoate Proteins 0.000 title claims abstract description 54
- 229960004824 triptorelin Drugs 0.000 title claims abstract description 54
- 238000000034 method Methods 0.000 title claims abstract description 22
- 238000001514 detection method Methods 0.000 claims abstract description 22
- 238000004811 liquid chromatography Methods 0.000 claims abstract description 14
- 238000009472 formulation Methods 0.000 claims abstract description 7
- 239000000203 mixture Substances 0.000 claims abstract description 7
- 238000003859 hyphenated technique Methods 0.000 claims abstract description 5
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 83
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 39
- 239000012071 phase Substances 0.000 claims description 20
- 239000007791 liquid phase Substances 0.000 claims description 15
- 238000002347 injection Methods 0.000 claims description 13
- 239000007924 injection Substances 0.000 claims description 13
- 239000002253 acid Substances 0.000 claims description 11
- 238000010828 elution Methods 0.000 claims description 11
- 238000002552 multiple reaction monitoring Methods 0.000 claims description 11
- 239000007921 spray Substances 0.000 claims description 9
- 238000006243 chemical reaction Methods 0.000 claims description 8
- 239000011260 aqueous acid Substances 0.000 claims description 7
- 238000012113 quantitative test Methods 0.000 claims description 7
- 241000124008 Mammalia Species 0.000 claims description 2
- 230000004060 metabolic process Effects 0.000 claims description 2
- 238000011160 research Methods 0.000 claims description 2
- 239000000523 sample Substances 0.000 abstract description 35
- 238000003556 assay Methods 0.000 abstract description 2
- 239000012472 biological sample Substances 0.000 abstract description 2
- 238000004949 mass spectrometry Methods 0.000 description 13
- 210000002381 plasma Anatomy 0.000 description 10
- 239000000243 solution Substances 0.000 description 10
- XBDQKXXYIPTUBI-UHFFFAOYSA-N dimethylselenoniopropionate Natural products CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 description 8
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 8
- 230000004044 response Effects 0.000 description 8
- 239000007864 aqueous solution Substances 0.000 description 7
- 238000002360 preparation method Methods 0.000 description 7
- 239000012086 standard solution Substances 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 241000282472 Canis lupus familiaris Species 0.000 description 6
- 101000857870 Squalus acanthias Gonadoliberin Proteins 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 229940088597 hormone Drugs 0.000 description 5
- 239000005556 hormone Substances 0.000 description 5
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 4
- 241001251200 Agelas Species 0.000 description 4
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 235000019253 formic acid Nutrition 0.000 description 4
- 238000001802 infusion Methods 0.000 description 4
- 235000019260 propionic acid Nutrition 0.000 description 4
- IUVKMZGDUIUOCP-BTNSXGMBSA-N quinbolone Chemical compound O([C@H]1CC[C@H]2[C@H]3[C@@H]([C@]4(C=CC(=O)C=C4CC3)C)CC[C@@]21C)C1=CCCC1 IUVKMZGDUIUOCP-BTNSXGMBSA-N 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- TZIHFWKZFHZASV-UHFFFAOYSA-N methyl formate Chemical group COC=O TZIHFWKZFHZASV-UHFFFAOYSA-N 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- DEQANNDTNATYII-OULOTJBUSA-N (4r,7s,10s,13r,16s,19r)-10-(4-aminobutyl)-19-[[(2r)-2-amino-3-phenylpropanoyl]amino]-16-benzyl-n-[(2r,3r)-1,3-dihydroxybutan-2-yl]-7-[(1r)-1-hydroxyethyl]-13-(1h-indol-3-ylmethyl)-6,9,12,15,18-pentaoxo-1,2-dithia-5,8,11,14,17-pentazacycloicosane-4-carboxa Chemical compound C([C@@H](N)C(=O)N[C@H]1CSSC[C@H](NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](CC=2C3=CC=CC=C3NC=2)NC(=O)[C@H](CC=2C=CC=CC=2)NC1=O)C(=O)N[C@H](CO)[C@H](O)C)C1=CC=CC=C1 DEQANNDTNATYII-OULOTJBUSA-N 0.000 description 2
- 239000000579 Gonadotropin-Releasing Hormone Substances 0.000 description 2
- 206010062767 Hypophysitis Diseases 0.000 description 2
- 108010016076 Octreotide Proteins 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003480 eluent Substances 0.000 description 2
- HQVFCQRVQFYGRJ-UHFFFAOYSA-N formic acid;hydrate Chemical compound O.OC=O HQVFCQRVQFYGRJ-UHFFFAOYSA-N 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 210000004907 gland Anatomy 0.000 description 2
- 239000003163 gonadal steroid hormone Substances 0.000 description 2
- XLXSAKCOAKORKW-AQJXLSMYSA-N gonadorelin Chemical compound C([C@@H](C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N1[C@@H](CCC1)C(=O)NCC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@H]1NC(=O)CC1)C1=CC=C(O)C=C1 XLXSAKCOAKORKW-AQJXLSMYSA-N 0.000 description 2
- 229940035638 gonadotropin-releasing hormone Drugs 0.000 description 2
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 2
- 238000002414 normal-phase solid-phase extraction Methods 0.000 description 2
- 229960002700 octreotide Drugs 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 230000001568 sexual effect Effects 0.000 description 2
- 230000000638 stimulation Effects 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- 102000009151 Luteinizing Hormone Human genes 0.000 description 1
- 108010073521 Luteinizing Hormone Proteins 0.000 description 1
- 206010060862 Prostate cancer Diseases 0.000 description 1
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- 239000006035 Tryptophane Substances 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 239000000556 agonist Substances 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 230000037058 blood plasma level Effects 0.000 description 1
- 238000010241 blood sampling Methods 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000007857 degradation product Substances 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000036267 drug metabolism Effects 0.000 description 1
- 229940011871 estrogen Drugs 0.000 description 1
- 239000000262 estrogen Substances 0.000 description 1
- 210000003608 fece Anatomy 0.000 description 1
- 230000003325 follicular Effects 0.000 description 1
- 235000013905 glycine and its sodium salt Nutrition 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 201000010260 leiomyoma Diseases 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 239000011806 microball Substances 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 210000003635 pituitary gland Anatomy 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 230000002028 premature Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000036279 refractory period Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 229960004799 tryptophan Drugs 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
Landscapes
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The present invention relates to a kind of method measuring trace Triptorelin, adopt liquid chromatography and mass spectrometric hyphenated technique to measure trace Triptorelin in sample, the method is simple to operate, result is accurate, specificity is good, highly sensitive.The invention still further relates to the content of described assay method for the Triptorelin in qualitative or quantitative detection biological sample and the purposes of triptorelin formulations pharmacokinetic thereof.
Description
Technical field
The present invention relates to a kind of method adopting liquid chromatography and mass Spectrometry for Determination trace Triptorelin.
Background technology
Triptorelin is the decapeptide of Prof. Du Yucang, is the analog of gonadotropin-releasing hormone (GRH) (GnRH).The improvement of its structure is by the Aminosteril KE (glycocoll) of the 6th in natural molecular structures, replaces with dextrorotation tryptophane.Triptorelin effect is identical with GnRH, but its extended plasma half-life and stronger to the affinity of GnRH acceptor, therefore Triptorelin becomes the robust agonist of GnRH acceptor.After Triptorelin injection, most first meeting Stimulation of Pituitary Gland secretion promoting sexual gland hormone (Gn), i.e. lutropin (LH) and follicular stimulating hormone (FSH).When hypophysis can enter refractory period after long-term stimulation, the release of promoting sexual gland hormone can reduce, and thus makes sex steroid (stosterone or estrogen) be reduced to castrate levels.Above-mentioned effect is reversible.Triptorelin is used to treat various hormone-dependent diseases as prostate cancer, mullerianosis, fibroid and sex premature clinically.
The product that Triptorelin has gone on the market mainly contains injection and long-acting slow-release microball preparation, the clinical common dose of Triptorelin: hypodermic injection once a day 0.5 milligram, continuous seven days, then hypodermic injection once a day 0.1 milligram, as maintenance dose; Inseminatio externalis art (IVF) hypodermic injection once a day 0.5 milligram, seven to ten days, then hypodermic injection once a day 0.1 milligram.The bad reaction of Triptorelin clinical practice is due to the suppressed and pharmacology spinoff that may cause of the generation of hormone, therefore should closely monitor sex steroid blood plasma level during clinical treatment.The clinical extremely low consumption of Triptorelin, just may cause extremely strong clinical adverse, these situations are all a kind of simple to operate in the urgent need to setting up, good and the high-sensitive trace Triptorelin detection technique of specificity, in the biological samples such as current blood plasma and excreta, the detection of trace Triptorelin utilizes radioimmunology to measure, what measure due to its is the specific fragment of polypeptide, in sample, other have the auto-degradation product of the metabolism fragment of antigenic determinant or Triptorelin also can react with immunoreagent, increase resultant error, and it has radioactivity, potential injury possibility is had to testing staff, and its kit is very expensive, senior instrument and equipment must be equipped with, be not suitable for clinical and scientific research use.
Summary of the invention
In view of the foregoing defects the prior art has, the present inventor furthers investigate, provide a kind of simple to operate, result is accurate, specificity good, the assay method of highly sensitive trace Triptorelin.Described method adopts liquid chromatography and mass spectrometric hyphenated technique, and the mobile phase of its efficient liquid phase adopts and carries out gradient elution containing aqueous acid-methanol system, and described acid is acetic acid or propionic acid, is preferably acetic acid; The concentration expressed in percentage by volume 0.01-0.1% of acid, is preferably 0.01-0.05%, is more preferably 0.02%.Described concentration expressed in percentage by volume is volume (ml) number of contained acid in 100ml water.
In LC-MS method, the selection of liquid chromatogram mobile phase has very important effect.The organic phase of mobile phase of the present invention selects methyl alcohol, compares other organic solvents such as acetonitrile, and methyl alcohol has higher response and good chromatographic behavior when measuring trace Triptorelin; Add the sensitivity that acid can significantly improve method in the aqueous phase of mobile phase, wherein interpolation acetic acid or propionic acid are than other acid such as formic acid, can significantly improve detectability nearly ten times.Described detectability refers to the least concentration (amount) of the tested component that can detect in the sample to which, namely produce signal (peak height) for sample concentration during baseline noise standard deviation k times, generally detectability is defined as concentration during signal to noise ratio (S/N ratio) (S/N) 3: 1.
Described gradient elution is containing the volume ratio table specific as follows of aqueous acid and methyl alcohol, and wherein the 3rd time period can omit, but preferably containing the 3rd time period, can reduce the high and noise of continuous sample introduction rear pillar voltage rise.
The method that the present invention also relates on the other hand described mensuration trace Triptorelin is for detecting the mammal content of Triptorelin and purposes of change procedure thereof in body after use triptorelin formulations.Successfully can be detected the trace Triptorelin in sample by method of the present invention, realize, after use triptorelin formulations, detecting content and the change procedure thereof of Triptorelin in its body.
The method of the invention is as a kind of trace Triptorelin LC-MS determination method, there is the sensitivity and specificity that are better than existing detection technique, can be used for the detection of trace Triptorelin in sample, for the detection of trace Triptorelin sample, determine that triptorelin formulations drug metabolism study in vivo provides effective detection means.The method of the invention can be used for the exploitation of triptorelin formulations, measuring the change using Triptorelin patient vivo medicine concentration clinically, can provide experimental basis by its dynamic change of Continuous Observation for instructing the reasonable employment of clinical Triptorelin.
Accompanying drawing explanation
Fig. 1 0.005ng/mL Triptorelin standard solution is the liquid quality detection collection of illustrative plates of 0.02% acetic acid aqueous solution-methyl alcohol at mobile phase
The average Drug-time curve collection of illustrative plates of Fig. 2 Triptorelin microballoon in Dog Plasma
Embodiment
Below by embodiment to the present invention's further instruction in addition, but do not limit the present invention in any form.
Embodiment 1 mobile phase is that 0.02% acetic acid aqueous solution-methyl alcohol detects Triptorelin
The preparation precision of standard serial solution sample takes 25mg Triptorelin standard items, puts in 25ml volumetric flask, adds methyl alcohol: water: formic acid (80: 20: 0.04) solubilize, and is diluted to scale, is mixed with the storing solution of 1mg/ml.Get Triptorelin storing solution appropriate, use methyl alcohol respectively: water: formic acid (60: 40: 0.08) is diluted to 0.005,0.01,0.03,0.1,0.3,1.0,3.0,10.0ng/ml.
Liquid-phase condition chromatographic column:
venusil MP-C18 column(2.1mm x 50mm, 3 μm, Agela).Mobile phase: A:0.02% acetic acid aqueous solution, B: methyl alcohol; Flow velocity: 0.6mL/min, column temperature: 40 DEG C, sample size: 10 μ l.Agilent1290 highly effective liquid phase chromatographic system, comprises binary infusion pump, automatic sampler, column oven.Gradient condition is as follows:
| Time(min) | A | B |
| 0 | 90 | 10 |
| 1 | 90 | 10 |
| 2.3 | 40 | 60 |
| 2.7 | 40 | 60 |
| 2.8 | 90 | 10 |
| 4.5 | 90 | 10 |
Mass Spectrometry Conditions: QTRAP5500 type mass spectrometer, is furnished with ion spray ionisation source, Applied Biosystem company of the U.S.
Ion gun: electron spray ionisation source (ESI), CAD:9, gas curtain atmospheric pressure (CUR): 35psi, GS1:55, GS2:55, TEM:575 DEG C; Injection electric: 5500V, positive ion mode detects; Scan mode is multiple-reaction monitoring (MRM) for the ionic reaction of quantitative test is m/z 656.5 → 249.1 and m/z 656.5 → 110.1; Separating bunch voltage DP voltage is 50V, and collision energy CE is respectively 42eV and 90eV.
With this liquid chromatography and Mass Spectrometry Conditions to standard solution sample detection, concentration is 0.005ng/ml sample, and detection response is 96cps, signal to noise ratio (S/N ratio) 6.0, and chromatogram is shown in accompanying drawing 1.
Embodiment 2 mobile phase is that 0.02% acetic acid aqueous solution-methyl alcohol detects Triptorelin
The preparation of standard serial solution sample is with embodiment 1
Liquid-phase condition chromatographic column:
venusil MP-C18 column(2.1mm x 50mm, 3 μm, Agela).Mobile phase: A:0.02% acetic acid aqueous solution, B: methyl alcohol; Flow velocity: 0.6mL/min, column temperature: 40 DEG C, sample size: 10 μ l.Agilent1290 highly effective liquid phase chromatographic system, comprises binary infusion pump, automatic sampler, column oven.Gradient condition is as follows:
| Time(min) | A | B |
| 0 | 90 | 10 |
| 1 | 90 | 10 |
| 2.3 | 40 | 60 |
| 2.7 | 40 | 60 |
| 2.71 | 2 | 98 |
| 4 | 2 | 98 |
| 4.01 | 90 | 10 |
| 5.5 | 90 | 10 |
Mass Spectrometry Conditions: QTRAP5500 type mass spectrometer, is furnished with ion spray ionisation source, Applied Biosystem company of the U.S.
Ion gun: electron spray ionisation source (ESI), CAD:9, gas curtain atmospheric pressure (CUR): 35psi, GS1:55, GS2:55, TEM:575 DEG C; Injection electric: 5500V, positive ion mode detects; Scan mode is multiple-reaction monitoring (MRM) for the ionic reaction of quantitative test is m/z 656.5 → 249.1 and m/z 656.5 → 110.1; DP is that 50V, CE are respectively 42eV and 90eV.
With this liquid chromatography and Mass Spectrometry Conditions to standard solution sample detection, concentration is 0.005ng/ml sample, and detection response is 93cps, and signal to noise ratio (S/N ratio) is 5.6.
Embodiment 3 mobile phase is that 0.05% acetic acid aqueous solution-methyl alcohol detects Triptorelin
The preparation of standard serial solution sample is with embodiment 1
Liquid-phase condition chromatographic column:
venusil MP-C18 column(2.1mm x 50mm, 3 μm, Agela).Mobile phase: A:0.05% acetic acid aqueous solution, B: methyl alcohol; Flow velocity: 0.4mL/min, column temperature: 30 DEG C, sample size: 10 μ l.Agilent 1290 highly effective liquid phase chromatographic system, comprises binary infusion pump, automatic sampler, column oven.Gradient condition is as follows:
| Time(min) | A | B |
| 0 | 90 | 10 |
| 1 | 90 | 10 |
| 4.0 | 50 | 50 |
| 5.0 | 50 | 50 |
| 5.1 | 5 | 95 |
| 6.5 | 5 | 95 |
| 7.0 | 90 | 10 |
| 9.0 | 90 | 10 |
Mass Spectrometry Conditions: QTRAP5500 type mass spectrometer, is furnished with ion spray ionisation source, Applied Biosystem company of the U.S.
Ion gun: electron spray ionisation source (ESI), CAD:9, gas curtain atmospheric pressure (CUR): 35psi, GS1:55, GS2:55, TEM:600 DEG C; Injection electric: 5000V, positive ion mode detects; Scan mode is multiple-reaction monitoring (MRM) for the ionic reaction of quantitative test is m/z 656.5 → 249.1 and m/z 656.5 → 110.1; DP is that 48V, CE are respectively 38eV and 88eV.
With this liquid chromatography and Mass Spectrometry Conditions to standard solution sample detection, concentration is 0.005ng/ml sample, and detection response is 86cps, and signal to noise ratio (S/N ratio) is 4.5.
Embodiment 4 mobile phase is that 0.1% propionic acid aqueous solution-methyl alcohol detects Triptorelin
The preparation of standard serial solution sample is with embodiment 1
Liquid-phase condition chromatographic column:
venusil MP-C18 column(2.1mm x 50mm, 3 μm, Agela).Mobile phase: A:0.1% propionic acid aqueous solution, B: methyl alcohol; Flow velocity: 0.6mL/min, column temperature: 50 DEG C, sample size: 10 μ l.Agilent 1290 highly effective liquid phase chromatographic system, comprises binary infusion pump, automatic sampler, column oven.Gradient condition is as follows:
| Time(min) | A | B |
| 0 | 95 | 5 |
| 1 | 95 | 5 |
| 3.0 | 30 | 70 |
| 3.5 | 30 | 70 |
| 3.51 | 2 | 98 |
| 4.0 | 2 | 98 |
| 4.01 | 95 | 5 |
| 5.5 | 95 | 5 |
Mass Spectrometry Conditions: QTRAP5500 type mass spectrometer, is furnished with ion spray ionisation source, Applied Biosystem company of the U.S.
Ion gun: electron spray ionisation source (ESI), CAD:9, gas curtain atmospheric pressure (CUR): 35psi, GS1:55, GS2:55, TEM:500 DEG C; Injection electric: 5000V, positive ion mode detects; Scan mode is multiple-reaction monitoring (MRM) for the ionic reaction of quantitative test is m/z 656.5 → 249.1 and m/z 656.5 → 110.1; DP is that 51V, CE are respectively 40eV and 88eV.
With this liquid chromatography and Mass Spectrometry Conditions to standard solution sample detection, concentration is 0.005ng/ml sample, and detection response is 74cps, signal to noise ratio (S/N ratio) 3.7.
Embodiment 5 mobile phase is that 0.02% aqueous formic acid-methyl alcohol detects Triptorelin
Standard serial solution sample, increases concentration 0.05ng/ml sample simultaneously, compound method with embodiment 1,
Liquid chromatogram mobile phase: 0.02% aqueous formic acid A-methyl alcohol B, other liquid chromatography and Mass Spectrometry Conditions are with embodiment 1
With this liquid chromatography and Mass Spectrometry Conditions to standard solution sample detection, concentration is 0.05ng/ml sample, and detection response is 180cps, signal to noise ratio (S/N ratio) 3.2.
Embodiment 6 mobile phase is that 0.01% trifluoroacetic acid aqueous solution-methyl alcohol detects Triptorelin
Embodiment 1 is shown in the preparation of standard serial solution sample
Liquid chromatogram mobile phase: 0.01% trifluoroacetic acid aqueous solution A-methyl alcohol B, other liquid chromatography and Mass Spectrometry Conditions are with embodiment 1
With this liquid chromatography and Mass Spectrometry Conditions to standard solution sample detection, concentration is 0.03ng/ml sample, and detection response is 98cps, signal to noise ratio (S/N ratio) 2.9.
Test example 1 detects Triptorelin in Dog Plasma
Trial drug: Triptorelin microballoon (Diphereline
, IPSEN company produces, lot number: C16453)
Experimental animal: Beagle dog 5, body weight is at about 12 ± 0.5kg, male
Test method: 5 Beagle dog administered intramuscular, dosage 0.3mg/kg gives, and administration volume is 0.15mL/kg; Respectively at 1h, 6h, 1d, 2d, 3d, 4d, 6d, 9d, 11d, 14d, 16d, 19d, 23d, 26d and 30d blood sampling after (0 hour) before administration and administration.
The preparation of typical curve: get 100 μ L Triptorelin standard items (0.01 respectively, 0.03, 0.1, 0.3, 1, 3, 10ng/mL), get the eppendorff pipe that 100 μ L dog blank plasmas are placed in 1.5mL, be configured to respectively be equivalent to 0.01, 0.03, 0.1, 0.3, 1, 3, the plasma solutions of 10ng/mL, add mark Octreotide 10ng/mL in 100 μ L, 500 μ L methyl alcohol, vortex 2min, the centrifugal 10min of 15000rpm, supernatant is added in the aqueous solution containing 600 μ L, after mixing, (activate on above-mentioned sample through 1mL methyl alcohol to WatersOasis HLB solid-phase extraction column, 1mL water balance) (1cc/30mg), then through 1mL methanol/water (60: 40) drip washing, 1% formic acid methyl alcohol collects eluent, in 60 DEG C of water-baths, pressurized air dries up, add 100 μ L methyl alcohol: 0.2% formic acid water (60: 40) vortex 2min redissolves, to be measured.
Plasma sample process: get 100 μ L Triptorelins standard items (Nat'l Pharmaceutical & Biological Products Control Institute) respectively: 0.005, 0.01, 0.03, 0.1, 0.3, 1, 3, 10ng/mL, get the eppendorff pipe that 100 μ L dog blank plasmas are placed in 1.5mL, be configured to respectively be equivalent to 0.01, 0.03, 0.1, 0.3, 1, 3, the plasma solutions of 10ng/mL, add mark Octreotide 10ng/mL in 100 μ L, 500 μ L methyl alcohol, vortex 2min, the centrifugal 10min of 15000rpm, supernatant is added in the aqueous solution containing 600 μ L, after mixing, (activate on above-mentioned sample through 1mL methyl alcohol to Waters Oasis HLB solid-phase extraction column, 1mL water balance) (1cc/30mg), then through 1mL methanol/water (60: 40) drip washing, 1% formic acid methyl alcohol collects eluent, in 60 DEG C of water-baths, pressurized air dries up, add 100 μ L methyl alcohol: 0.2% formic acid water (60: 40) vortex 2min gets 10 μ l sample introductions to liquid matter system.
Liquid phase-mass spectrum Series detectors condition is with embodiment 2
Test findings: accompanying drawing 2 is shown in by pharmacokinetics collection of illustrative plates
Result shows: detection method of the present invention, and after use triptorelin formulations, in the whole medication cycle, detect trace Triptorelin in blood plasma and have better response, data meet the requirement of LLOQ.
LLOQ is lower limit of quantification, i.e. lower limit of quantitation (minimum quantitative limit).FDA specifies the S/N > 5 of LLOQ point and the CV < 20%, S/N of LLOQ point and signal to noise ratio (S/N ratio), and CV is the coefficient of variation, and it equals standard deviation/mean value.
Claims (5)
1. one kind measures the method for trace Triptorelin, it is characterized in that adopting liquid chromatography and mass spectrometric hyphenated technique, the mobile phase of efficient liquid phase adopts methyl alcohol B-to carry out gradient elution containing aqueous acid A system, described acid is acetic acid, the concentration expressed in percentage by volume of acid is 0.02%, and the chromatographic column of liquid-phase condition is Venusil MP-C18 column, and specification is 2.1mm x 50mm, 3 μm, column temperature 40 DEG C; Flow velocity 0.6mL/min; Type of elution is gradient elution, is specially: 0-1 minute, and the volume ratio containing aqueous acid A and methyl alcohol B is 90: 10; The volume ratio of 2.3-2.7 minute, A and B is 40: 60; 2.8-4.5 minute, A and B volume ratio is 90: 10; Mass spectrographic injection electric is 5500V, and positive ion mode detects; Scan mode is multiple-reaction monitoring, and the ionic reaction for quantitative test is m/z 656.5 → 249.1 and m/z 656.5 → 110.1; Separating bunch voltage DP voltage is 50V, and collision energy CE is respectively 42eV and 90eV.
2. one kind measures the method for trace Triptorelin, it is characterized in that adopting liquid chromatography and mass spectrometric hyphenated technique, the mobile phase of efficient liquid phase adopts methyl alcohol B-to carry out gradient elution containing aqueous acid A system, described acid is acetic acid, the concentration expressed in percentage by volume of acid is 0.02%, and the chromatographic column of liquid-phase condition is Venusil MP-C18 column, and specification is 2.1mm x 50mm, 3 μm, column temperature 40 DEG C; Flow velocity 0.6mL/min; Type of elution is gradient elution, is specially: 0-1 minute, and the volume ratio containing aqueous acid A and methyl alcohol B is 90: 10; The volume ratio of 2.3-2.7 minute, A and B is 40: 60; The volume ratio of 2.71-4 minute, A and B is 2: 98; 4.01-5.5 minute, A and B volume ratio is 90: 10; Mass spectrographic injection electric is 5500V, and positive ion mode detects; Scan mode is multiple-reaction monitoring, and the ionic reaction for quantitative test is m/z 656.5 → 249.1 and m/z 656.5 → 110.1; Separating bunch voltage DP voltage is 50V, and collision energy CE is respectively 42eV and 90eV.
3. one kind measures the method for trace Triptorelin, it is characterized in that adopting liquid chromatography and mass spectrometric hyphenated technique, the mobile phase of efficient liquid phase adopts methyl alcohol B-to carry out gradient elution containing aqueous acid A system, described acid is acetic acid, the concentration expressed in percentage by volume of acid is 0.05%, and the chromatographic column of liquid-phase condition is Venusil MP-C18 column, and specification is 2.1mm x 50mm, 3 μm, column temperature 30 DEG C; Flow velocity 0.4mL/min; Type of elution is gradient elution, is specially: 0-1 minute, and the volume ratio containing aqueous acid A and methyl alcohol B is 90: 10; The volume ratio of 4.0-5.0 minute, A and B is 50: 50; The volume ratio of 5.1-6.5 minute, A and B is 5: 95; 7.0-9.0 minute, A and B volume ratio is 90: 10; Mass spectrographic injection electric is 5000V, and positive ion mode detects; Scan mode is multiple-reaction monitoring, and the ionic reaction for quantitative test is m/z 656.5 → 249.1 and m/z 656.5 → 110.1; Separating bunch voltage DP voltage is 48V, and collision energy CE is respectively 38eV and 88eV.
4. according to the arbitrary described method of claim 1-2, it is characterized in that mass spectrographic ion gun is electron spray ionisation source, CAD is 9, and gas curtain atmospheric pressure is 35psi, GS1 be 55, GS2 be 55, TEM is 575 DEG C.
5. method according to claim 2, for mammal after use triptorelin formulations, the content of Triptorelin and the purposes of metabolism research in detection bodies.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110409108.2A CN103163258B (en) | 2011-12-09 | 2011-12-09 | A kind of method measuring trace Triptorelin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110409108.2A CN103163258B (en) | 2011-12-09 | 2011-12-09 | A kind of method measuring trace Triptorelin |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103163258A CN103163258A (en) | 2013-06-19 |
| CN103163258B true CN103163258B (en) | 2015-09-23 |
Family
ID=48586517
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201110409108.2A Active CN103163258B (en) | 2011-12-09 | 2011-12-09 | A kind of method measuring trace Triptorelin |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103163258B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104458929B (en) * | 2013-09-24 | 2016-12-07 | 山东绿叶制药有限公司 | A kind of assay method detecting gonadotropin releasing hormone analogues and testosterone |
| CN108802227A (en) * | 2018-06-19 | 2018-11-13 | 大连工业大学 | The joint identification method of biologically active polypeptide sequence |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AR012448A1 (en) * | 1997-04-18 | 2000-10-18 | Ipsen Pharma Biotech | COMPOSITION IN THE FORM OF MICROCAPSULES OR IMPLANTS COMPRISING A BIODEGRADABLE CONTAINER, POLYMER OR CO-POLYMER, OR A MIXTURE OF SUCH EXCIPIENTS, AND AN ACTIVE SUBSTANCE OR MIXTURE OF ACTIVE SUBSTANCES, PROCEDURE FOR THE PREPARATION OF A SUBSTANCE IN A SUBSTANCE |
| ATE452145T1 (en) * | 2005-05-03 | 2010-01-15 | Novetide Ltd | METHOD FOR PRODUCING PEPTIDE DERIVATIVES |
| CN101357936B (en) * | 2007-07-31 | 2013-07-03 | 崔颀 | Method for synthesizing triptorelin from solid phase polypeptide |
| WO2009114959A1 (en) * | 2008-03-20 | 2009-09-24 | 中国人民解放军军事医学科学院毒物药物研究所 | Injectalble sustained-release pharmaceutical formulation and method for preparing it |
| CN102048699B (en) * | 2009-11-03 | 2012-11-07 | 长春金赛药业有限责任公司 | Preparation method for sustained release microsphere of injection triptorelin acetate |
| CN102125517B (en) * | 2011-02-12 | 2013-12-11 | 成都师创生物医药科技有限公司 | Application of low-concentration vesicular phospholipid gel as slow release carrier for small-molecule peptide drug |
-
2011
- 2011-12-09 CN CN201110409108.2A patent/CN103163258B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN103163258A (en) | 2013-06-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104807921A (en) | Method for detecting 10 kinds of steroid hormones in serum through high performance liquid chromatography tandem mass spectrum technique | |
| Ma et al. | Simultaneous determination of tetrahydropalmatine, protopine, and palmatine in rat plasma by LC-ESI-MS and its application to a pharmacokinetic study | |
| CN110082440A (en) | The method of the ultra performance liquid chromatography tandem mass spectrum measurement molecular targeted concentration of blood plasma | |
| CN103454360A (en) | Ultrafiltration and UPLC-MS/MS (ultra-high performance liquid chromatography tandem mass spectrometry) method for measuring concentration of free docetaxel in human plasma | |
| CN111766312A (en) | Method for detecting antifungal drugs in serum by ultra-high performance liquid chromatography tandem mass spectrometry technology | |
| Sheng et al. | Pharmacokinetic and excretion study of three secoiridoid glycosides and three flavonoid glycosides in rat by LC–MS/MS after oral administration of the Swertia pseudochinensis extract | |
| WO2022110569A1 (en) | Lc-ms/ms measurement method for aloesin in rat blood plasma | |
| Handelsman et al. | Detection and effects on serum and urine steroid and LH of repeated GnRH analog (leuprolide) stimulation | |
| Liu et al. | Development and validation of a sensitive LC-MS/MS method for simultaneous quantification of thirteen steroid hormones in human serum and its application to the study of type 2 diabetes mellitus | |
| CN110045048A (en) | A kind of HPLC-MSMS method of two kinds of anti-tumor drug concentration in measurement human plasma | |
| CN103163258B (en) | A kind of method measuring trace Triptorelin | |
| CN103529135B (en) | The analysis method of diazepam and cylinder metabolism-ure thereof in LC-MS/MS method detection people's urine sample | |
| Huang et al. | Simultaneous determination of human plasma protein binding of bioactive flavonoids in Polygonum orientale by equilibrium dialysis combined with UPLC–MS/MS | |
| CN107014915A (en) | The quantitative detecting method of abiraterone in whole blood | |
| Han et al. | An analytical strategy to characterize the pharmacokinetics and pharmacodynamics of triptorelin in rats based on simultaneous LC–MS/MS analysis of triptorelin and endogenous testosterone in rat plasma | |
| Sun et al. | Pharmacokinetic study of zhebeirine in mouse blood by ultra-performance liquid chromatography/tandem mass spectrometry | |
| CN103575820B (en) | The analysis method of 5 kinds of flavonoid glycosides and application in pharmacokinetics thereof in blood plasma | |
| CN103940918A (en) | A method of simultaneously detecting the content of artesunate and the content of dihydroartemisinin in animal blood plasma | |
| CN107167540B (en) | The method for measuring the DTPA-Zn in human urine biological sample | |
| Cao et al. | Study of the determination and pharmacokinetics of bufadienolides in dog's plasma after administration of Liu-Shen-Wan by high performance liquid chromatography time-of-flight mass spectrometry | |
| CN104458929B (en) | A kind of assay method detecting gonadotropin releasing hormone analogues and testosterone | |
| CN107045031B (en) | The LC-MS/MS high-flux detection method of saxagliptin and 5- hydroxyl saxagliptin in human plasma | |
| Lee et al. | Development of new clean-up method for UPLC–MS/MS analysis of leuprolide | |
| CN111830154B (en) | Separation method and application of oxytocin and 8 epimers thereof | |
| CN111812240B (en) | Separation method and application of oxytocin and three impurities |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C53 | Correction of patent of invention or patent application | ||
| CB02 | Change of applicant information |
Address after: 264003 Yantai hi tech Zone, Shandong venture Road No. 15 Applicant after: SHANDONG LUYE PHARMACEUTICAL Co.,Ltd. Address before: 264003 Laishan, Shandong Province Bao Road, No. 9 District, No. Applicant before: SHANDONG LUYE PHARMACEUTICAL Co.,Ltd. |
|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20220704 Address after: 264003 No. 368, Changning Road, Laishan District, Yantai City, Shandong Province Patentee after: Shandong nuoqiao Biotechnology Co.,Ltd. Address before: 264003 No. 15, Chuangye Road, high tech Zone, Yantai City, Shandong Province Patentee before: SHANDONG LUYE PHARMACEUTICAL Co.,Ltd. |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20230414 Address after: 611139 Chengdu Tianfu International Biological City, Chengdu, Sichuan Province (No. 618, Fenghuang Road, Shuangliu District) Patentee after: Nuoqiao Pharmaceutical (Chengdu) Co.,Ltd. Address before: 264003 No. 368, Changning Road, Laishan District, Yantai City, Shandong Province Patentee before: Shandong nuoqiao Biotechnology Co.,Ltd. |
|
| TR01 | Transfer of patent right |