CN102735764A

CN102735764A - Method for determining content of ribavirin in blood plasma

Info

Publication number: CN102735764A
Application number: CN2011100815719A
Authority: CN
Inventors: 吴飞; 丁黎
Original assignee: SHANGHAI ZHANGJIANG TRADITIONAL CHINESE MEDICINE MODERN PHARMACEUTICAL PREPARATION TECHNOLOGY ENGINEERING RESEARCH CENTER
Current assignee: SHANGHAI ZHANGJIANG TRADITIONAL CHINESE MEDICINE MODERN PHARMACEUTICAL PREPARATION TECHNOLOGY ENGINEERING RESEARCH CENTER
Priority date: 2011-03-31
Filing date: 2011-03-31
Publication date: 2012-10-17

Abstract

The invention relates to a method for determining the content of ribavirin in blood plasma, comprising the following steps of: (1) treatment of a blood plasma sample: adding an internal standard solution (6-methyl nicotinic acid) and methanol into drug-containing blood plasma containing a ribavirin component, scrolling, uniformly mixing, centrifuging, taking an upper clear solution, adding it into water bath at 35 DEG C, blow-drying by the use of nitrogen flow, redissolving by a mobile phase, scrolling, adding chloroform, scrolling and standing, centrifuging, and sucking the upper clear solution for measurement; (2) chromatographic conditions for a LC-MS method for detection: chromatographic column: Sepax HP-C18, dp 3 micon, 2.1*100mm, column temperature: 30 DEG C, mobile phase: 30mmol/L of an ammonium acetate aqueous solution (containing 0.2% of formic acid), flow velocity: 0.2 ml/min; mass spectrum condition: ion detection mode: selective ion monitoring (SIM), ion polarity: positive ion (Positive), ionizationoun mode: pneumatic auxiliary electro-spray ionizationoun (ESI), detection object: ribavirin ([M+Na]+, m/z 267.0); 6-methyl nicotinic acid (internal standard, [M+H]+, m/z 138.1). The invention is suitable for in vitro pharmacokinetics research of ribavirin in blood plasma.

Description

Ribavirin Determination on content method in a kind of blood plasma

Technical field

The invention belongs to biopharmaceutical analysis and pharmacokinetics field, particularly relate to Ribavirin Determination on content method in the blood plasma.

Background technology

Ribavirin is the strong antiviral drugs of imitating of wide spectrum, is single phosphoric acid time purine nucleosides dehydrogenase inhibitor, suppresses IMB and changes guanylic acid into, and it is synthetic to hinder viral nucleic acid, stops virus replication and propagation, thereby reaches antiviral effect.Multiple RNA and dna virus all there are the obvious suppression effect, HIV virus is also had stronger inhibiting effect.The Ribavirin few side effects; Adverse reaction rate is low; According to its pharmacological action; Will note heavy dose of long-term phenomenons such as leukopenia, anaemia, serum transaminase and cholerythrin rising that cause of using during use, for guaranteeing that clinical drug safety is effective, the Ribavirin Determination on content is of crucial importance in the blood plasma.Up to the present; Relevant Ribavirin Determination on content method is various; Such as ultraviolet spectroscopy, high performance liquid chromatography (HPLC), reversed-phased high performace liquid chromatographic (RP-HPLC), polarimetry, colourimetry, high performance capillary electrophoresis method (HPCE), linear-sweep polarography and periodate method etc., more rarely there is the LC-MS method to measure the report of Ribavirin content.

Summary of the invention

Technical matters to be solved by this invention provides a kind of new Ribavirin content assaying method; This method has higher specificity; Can accurately measure the concentration of Ribavirin in the blood plasma, sensitivity is higher, and control and clinical rational drug use provide foundation in order to ensure drug quality.

The technical matters that the present invention solves realizes through following technical scheme:

Ribavirin Determination on content method comprises the following steps: in the blood plasma

(1) plasma sample pre-service

A. the blood plasma that contains Ribavirin adds earlier inner mark solution (6-methylnicotinic acid) mixing, adds behind the methyl alcohol vortex mixing centrifugal again; Get the upper strata settled solution and in 35 ℃ of water-baths, dry up, redissolve, add chloroform behind the vortex with moving phase with nitrogen stream; It is centrifugal that vortex leaves standstill the back, draws the upper strata settled solution;

B. moving phase detects with the LC-MS method after redissolving;

(2) LC-MS detection method

Chromatographic condition: chromatographic column: Sepax HP-C18, dp 3 μ m, 2.1 * 100mm, column temperature: 30 ℃, moving phase: 30mmol/L ammonium acetate aqueous solution (containing 0.2% formic acid), flow velocity: 0.2ml/min; Mass spectrum condition: ion detection mode: selectivity ion detection (SIM), ion polarity: positive ion (Positive), ionization mode: pneumatic auxiliary electro-spray ionization (ESI).

Said step (2) gas helps electric spray ion source, adopts positive ion mode, and transmission voltage is 120V, and the dry gas flow velocity is 8L/min, and spray chamber pressure is 40psig, and the dry gas temperature is 350 ℃, and the detection ion that is used for quantitative test is respectively m/z 267.0 ([M+Na] ⁺, Ribavirin) and m/z 138.1 ([M+H] ⁺, interior mark 6-methylnicotinic acid).

One of characteristic of the present invention is to use chloroform as the reagent of removing albumen and endogenous impurity, can eliminate the endogenous impurity of measuring in the blood plasma and disturb.The present invention has the good recovery and precision, and is simple and efficient, do not receive the interference of other compositions in the preparation, meets the biological sample analysis requirement, but Ribavirin content in the sensitive determination human plasma is suitable for the Ribavirin pharmacokinetic.

The present invention guarantees that plasma sample measures the effective credible accurate of result;, the every batch of blood sample all adds at random when measuring and each 2 parts of the standard quality-control samples of basic, normal, high 3 concentration of replicate determination (20,200 and 1200 μ g/L); Test 12 altogether and analyzed the lot sample article; Each is analyzed batch Quality Control and measures the resultant error percent all less than ± 15%, meets the biopharmaceutical analysis requirement.

Description of drawings

The scanning of the mass spectrum figure of Fig. 1 Ribavirin.

The scanning of the mass spectrum figure of Figure 26-methylnicotinic acid.

The blank blood sample chromatogram of Fig. 3.

Fig. 4 Ribavirin standard items chromatogram.

Mark 6-methylnicotinic acid reference substance chromatogram in Fig. 5.

Embodiment:

Ribavirin content assaying method in the human plasma is in pastille blood plasma, to add earlier inner mark solution (6-methylnicotinic acid) mixing, adds behind the methyl alcohol vortex mixing centrifugal again; Get supernatant and in 35 ℃ of water-baths, dry up, redissolve, add chloroform behind the vortex with the moving phase of 30mmol/L ammonium acetate aqueous solution (containing 0.2% formic acid) with nitrogen stream; It is centrifugal that vortex leaves standstill the back, draws the upper strata settled solution, and promptly available LC-MS measures (chromatographic condition: chromatographic column: Sepax HP-C18; Dp 3 μ m, 2.1 * 100mm, column temperature: 30 ℃; Moving phase: 30mmol/L ammonium acetate aqueous solution (containing 0.2% formic acid), flow velocity: 0.2ml/min; Mass spectrum condition: ion detection mode: selectivity ion detection, ion polarity: positive ion, ionization mode: pneumatic auxiliary electro-spray ionization).

Measure the result: typical curve: get blank plasma 0.2ml; Add an amount of Ribavirin standard solution; Be mixed with and be equivalent to the solution that the Ribavirin PC is 10,30,100,300,600,1000 and 1500 μ g/L, according to the determination step operation, the record chromatogram; With the testing concentration is horizontal ordinate; The peak area ratio of determinand and internal standard compound is an ordinate; (weight coefficient w=1/C * C) least square method is carried out regressing calculation with weighting; Trying to achieve regression equation is: f=0.008268+0.1257 * C, r=0.9992, the minimum 10 μ g/L that quantitatively are limited to of Ribavirin in the blood plasma.Precision of the present invention: get blank plasma and add an amount of Ribavirin standard solution; Be made into the sample (20,200 and 1200 μ g/L) of basic, normal, high 3 concentration respectively; Press sample determination method replication 5 times in a few days, in a few days RSD is respectively 5.1%, 3.1% and 3.2%.With 3 concentration sample METHOD FOR CONTINUOUS DETERMINATION of method preparation 3 days, each concentration replication 5 times, RSD is respectively 5.5%, 5.5% and 3.3% in the daytime.Extraction recovery of the present invention: get blank plasma and add an amount of Ribavirin standard solution; Be made into the Ribavirin (20,200 and 1200 μ g/L) of basic, normal, high 3 concentration respectively; Press sample determination method replication 5 times; Ratio calculate recovery rate with measured value and theoretical value is respectively 81.4%, 78.0% and 82.3%, and average recovery rate is 80.6%.

Claims

1. Ribavirin Determination on content method in the blood plasma comprises the following steps:

(1) plasma sample pre-service

A. the pastille blood plasma that contains the Ribavirin composition; Add inner mark solution (6-methylnicotinic acid) vortex, add the methyl alcohol vortex mixing of 3-6 times of blood plasma volume again after, high speed centrifugation; Get the upper strata settled solution and in 30-70 ℃ of water-bath, dry up, redissolve with the moving phase of 1-2 times of blood plasma volume with nitrogen stream; Add the equal-volume chloroformic solution in the redissolution liquid, vortex leaves standstill the back high speed centrifugation, and it is to be measured to draw the upper strata settled solution.

B. the upper strata settled solution detects with the LC-MS method.

(2) LC-MS detection method:

2. the assay method of Ribavirin in the blood plasma according to claim 1; It is characterized in that said step (2) gas helps electric spray ion source, adopt positive ion mode, transmission voltage is 120V; The dry gas flow velocity is 8L/min; Spray chamber pressure is 40psig, and the dry gas temperature is 350 ℃, and the detection ion that is used for quantitative test is respectively m/z 267.0 ([M+Na] ⁺, Ribavirin) and m/z 138.1 ([M+H] ⁺, interior mark 6-methylnicotinic acid).

3. the assay method of Ribavirin in the blood plasma according to claim 1 is characterized in that using chloroform as the reagent of removing albumen and endogenous impurity, can eliminate the endogenous impurity of measuring in the blood plasma and disturb.