KR101003551B1 - Method of analyzing bisphophonate compound using derivative reaction - Google Patents

Abstract

The present invention relates to a method for measuring a bisphosphonate compound derivative, and more particularly to a method for measuring a bisphosphonate compound derivative using HPLC-MS / MS after derivatization of the bisphosphonate compound using trimethyloxyaryldiazomethane. It is about. In the clinical trials such as bioavailability test, bioequivalence test, and therapeutic effect monitoring of bisphosphonate compounds using the present invention, their concentration in plasma or urine concentration can be measured with high sensitivity.

Bisphosphonate compounds, trimethyloxyaryldiazomethane, risedronate

Description

Method of analyzing bisphophonate compound using derivative reaction

The present invention relates to a method for measuring a bisphosphonate compound using a derivatization reaction, and more specifically, after derivatizing the bisphosphonate compound using trimethyloxyaryl diazomethane, the bisphosphonate using HPLC-MS / MS. The present invention relates to a method for measuring a compound derivative.

In addition to aging population, osteoporosis patients also increase significantly every year. Risedronate (Figure 1 and Table 1) is a third generation bisphophonate compound developed by Procter & Gamble (P & G).

Recedronate has increased physicochemical stability and bone resorption inhibition effect by replacing P-O-P bond of pyrophosphate with P-C-P bond. Animal testing has shown that risedronate inhibits bone resorption more effectively than the first and second generation bisphosphonate compounds (see Ref. 1).

Bisphosphonate compounds with PCP structure are widely used in osteoporosis, Paget's disease, osteolytic metastasis (see Ref. 2, Ref. 3 and Ref. 4). . Therefore, bisphosphonates in biological fluids such as blood for the purpose of bioavailability (BA), bioequivalence (BE), and therapeutic drug monitoring (TDM) of bisphosphonate compounds. The method of measuring the concentration of) with high sensitivity is very important.

However, since most of the bisphosphonate compounds do not have chromophores, they are derivatized with 2,3-naphthalene dicarboxyaldehyde-5,6 or 9-fluorenyl-methylchloroformate-7 for UV or fluorescence detection and analyzed by HPLC. Is reported. In GC / MS or LC / MS, bisphosphonates were converted into volatile derivatives by acylation or sillation (see Ref. 8, Ref. 9 and Ref. 10). . However, most of these methods had a quantitative sensitivity of 1 ng / ml or more, and more sensitive assays were needed for use in clinical trials of bisphosphonate compounds.

On the other hand, Zhu et al. Derivatized risedronate in serum and urine using diazomethane as a derivatization reagent, and quantitative sensitivity of 0.2 ng / ml by LC-MS / MS method. Was developed (see Ref. 11). However, diazomethane is very toxic and highly explosive, so derivatization of bisphosphonate should be carried out in a safety cabinet, but it has a drawback that is difficult to carry out in the laboratory. In addition, diazomethane is banned in Korea due to its strong explosive properties.

An object of the present invention is to provide a method for derivatizing a bisphosphonate compound derivative using HPLC-MS / MS after derivatizing the bisphosphonate compound with trimethyloxyaryldiazomethane.

In order to achieve the above object, the present invention provides a method for derivatizing a bisphosphonate compound derivative using HPLC-MS / MS after derivatizing the bisphosphonate compound using trimethyloxyaryl diazomethane.

According to the present invention, when performing a clinical test such as bioavailability test (BA), bioequivalence test (BE), therapeutic effect monitoring (TDM), etc., bisphosphonate compounds are measured with high sensitivity in plasma or urine concentration. can do.

The present invention comprises the steps of derivatizing the bisphosphonate compounds (1) by adding trimethylsilyldiazomethane to the plasma sample (bisphosphonate compounds); (2) removing the unreacted material and the bisphosphonate compound through the solid phase extraction after the induction emulsification process; And injecting the sample obtained in (3) into LC-MS / MS to determine the bisphosphonate compound derivative.

In the above, bisphosphonate compounds are etidronate, clodronate, tiludronate, pamidronate, neridronate, olpadronate , Alendronate, albanronate, ibandronate, risedronate and zoleronate, characterized in that any one or more selected from the group consisting of (zoledronate).

In the above, it is preferable to use trimethylsilyldiazomethane alone or to mix trimethylsilyldiazomethane in an appropriate ratio such as other reagents, methanol, benzene and the like as shown below.

<TMS-DAM Solution>

Trimethylsilyldiazomethan: Metanol-1: 1 (v / v)

Trimethylsilyldiazomethan: Metanol-1: 2 (v / v)

Trimethylsilyldiazomethan: Metanol-1: 3 (v / v)

Trimethylsilyldiazomethan: Metanol: Benzen -1: 1: 1 (v / v / v)

Trimethylsilyldiazomethan: Metanol: Benzen -1: 2: 1 (v / v / v)

Trimethylsilyldiazomethan: Metanol: Benzen -1: 2: 2 (v / v / v)

Trimethylsilyldiazomethan: hexane (1: 3, v / v) / Metanol: Benzen (1: 1, v / v)

-(1/1, v / v)

Trimethylsilyldiazomethan: hexane (1: 3, v / v) / Metanol: Benzen (1: 1, v / v)

-(1/2, v / v)

Trimethylsilyldiazomethan: hexane (1: 3, v / v) / Metanol: Benzen (1: 1, v / v)

-(1/3, v / v)

Trimethylsilyldiazomethan: hexane (1: 3, v / v) / Metanol: Benzen (1: 1, v / v)

-(1/4, v / v)

In the above description, the solid column is preferably a SAX (eg, InertSep SAX) solid column, and a column having similar properties may be used.

The present invention derivatizes bisphosphonate with trimethylsilyldiazomethane, and then quantitates or quantifies it using an analyzer such as HPLC, GC-MS, LC-MS, and LC-MS / MS. It is desirable to make it through the process.

The method of the present invention can be applied to the determination of bisphosphonates in biological fluids. The biological fluid refers to components derived from living bodies such as blood, plasma, serum, urine, tissue fluid, bone marrow, and cerebrospinal fluid.

According to a preferred embodiment of the present invention, after derivatizing risedronate using risedronate, which is a representative compound of a bisphosphonate compound, trimethyloxyaryldiazomethane having no explosive properties as a derivatization reagent, LC-MS / MS method can be analyzed with high sensitivity of 0.2ng / ml. This method is applicable to the quantification of drugs with bisphosphonate structures.

The present invention was confirmed to provide sufficient sensitivity to conduct bioavailability studies after oral administration of risedronate, and there was no problem in obtaining the pharmacokinetic parameters of risedronate. Therefore, the method proposed in the present invention can be used to measure the concentration of bisphosphonates in biological fluids.

The present invention preferably uses zoledronate as an internal standard of risedronate.

In addition, the present invention is a high-sensitivity quantitative method, the reaction process can be safely carried out without a special device in a common laboratory, and is a suitable method for practical application, and because it has sufficient quantitative sensitivity, it is a bisphosphonate system including risedronate. It is expected to be useful for drug bioavailability test, bioequivalence test, drug treatment monitoring test, and clinical test.

The basic backbone of bisphosphonate is P-C-P bonds. Depending on which side chain (binding side) is attached to this, a compound having different drug efficacy and bone bonds is obtained. The chemical structure of bisphosphonate known to date is summarized in <Table 1>.

Table 1 Chemical Structure of Bisphosphonates

Compound R1 side chain R2 side chain Etidronate -OH -CH ₃ Clodronate -Cl -Cl Tiludronate -H ClS Pamidronate -OH NH ₂ Neridronate -OH NH ₂ (CH ₂ ) ₅ Olpadronate -OH CH ₃ CH ₃ N Alendronate -OH NH ₂ Ibandronate -OH CH ₃ (H ₂ C) ₄ CH ₃ N Risedronate -OH N Zoledronate -OH NN

1 schematically shows a common structure of bisphosphonates. The long side chain R2 is related to chemical properties, types of pharmacological action, the strength of the drug, etc. The short side chain R1, also called "hooks or hooks," affects chemical and pharmacokinetics. It is known to exert.

Meanwhile, the mechanism of the derivatization of bisphosphonates developed by Zhu et al. With diazomethane proceeds as shown in FIG. 2.

When Zhu et al. Derivatized risedronate with diazomethane, tetramethyl derivatives of risedronate were formed, and the derivative was well ionized in positive ESI mode. The result is a dramatic increase in sensitivity (see Ref. 11).

However, the diazomethane used by Zhu et al. Is highly toxic and highly explosive, making it a difficult reagent for general laboratories.

In the present invention, the same tetramethyl-bisphosphonate is obtained by using trimethylsilyldiazomethane, which is not explosive and can be treated like a general reagent in a general laboratory, as a derivatization reagent, and then these derivatives are converted into LC-MS / A high sensitivity assay of 0.2 ng / ml was developed by measuring using MS.

The method of the present invention,

Collecting blood from a donor to measure plasma concentrations of risedronate, and pretreating the plasma sample by pretreating the plasma obtained therefrom;

Derivatizing by adding a derivatization reagent to the plasma sample obtained in step 1);

After the derivatization process of step 2), removing the unreacted material and the derivatization reagent through solid column extraction; And

And injecting the sample obtained in step 3) into LC-MS / MS to measure the sample.

The pretreatment process of steps 1) to 3) was carried out as follows.

I) Samples for calibration curves, quality control samples and plasma specimens were prepared.

Ii) To 0.6 ml of each sample, 0.3 ml of internal standard (20 ng / ml) was added to the test tube.

Viii) 1.5 ml of water was added.

Viii) was added to an Inertsep SAX solid column.

Iii) washed with 2.0 ml of water.

V) washed with 2.0 ml methanol.

Viii) washed with 2.0 ml of Acetonitrile.

Viii) 45 ml of TMS-DAM solution [Trimethylsilyldiazomethan: hexane (1: 3, v / v) / Metanol: Benzen (1: 1, v / v)-(1/4, v / v)] It was left for 60 minutes at.

V) eluted with 2.0 ml of Methanol.

V) The eluate was concentrated to dryness at 50 ° C. under nitrogen gas.

V) Redissolved in 0.1 ml of 90% acetonitrile aqueous solution.

Iiiii) 5 μl was analyzed by LC-MS / MS.

LC-MS / MS measurement conditions of step iii) were as follows.

<LC conditions> Pump LC-20AD (Shimadzu) Degasser DGU-20A5 (Shimadzu) Autosampler SIL-20AC (Shimadzu) Column compartment CTO-10ASvp (Shimadzu) System controller CBM-20A (Shimadzu) Analytical column Atlantis HILIC, 3 μm, 2.1 mm I.D. x 50mm L. (Waters) Mobile phase A: 10 mmol / L Ammonium formate aq.
B: Acetonitrile
A: B = 10: 90 Column temperature 40 Sample cooler 4 Injection volume 5 μl Run time 5 min <Mass system> MS / MS API-4000QTRAP AB / MDS SCIEX Data processing Analyst (ver. 1.4.2)

Hereinafter, the contents of the present invention will be described in more detail with reference to Examples. However, these examples are only presented to understand the content of the present invention, but the scope of the present invention is not limited to these embodiments.

< Example 1 >

Create calibration curve

Lysedronate standard (LKT Laboratories Inc.) was dissolved in H ₂ O to make 100 ㎍ / ml as a free base, then refrigerated, and the solution was diluted with blank plasma that was stored in frozen plasma to give rise to the concentration of risedronate in plasma. Standard plasma samples were prepared so that they were 0.2, 0.5, 1, 2, 5, 10, 16 and 20 ng / ml, respectively. 0.6 ml of the standard plasma sample was mixed with 0.3 ml of internal standard solution (zoledronate, 20 ng / ml) and 1.5 ml of H ₂ O. The SAX cartridge (Inertsep) was conditioned with 3 ml of methanol and H ₂ O, respectively, and the samples were applied and washed with 2 ml of H ₂ O, methanol, and acetonitrile. Trimethylsilyldiazomethane reaction reagent [Trimethylsilyldiazomethan: hexane (1: 3, v / v) / Metanol: Benzen (1: 1, v / v)-(1/4, v / v)]] 1 ml Added and reacted for 60 minutes, and then eluted with 2 ml of methanol. The eluate was vacuum dried and redissolved in 90% acetonitrile solution (acetonitrile aq.) And 5 μl was injected into LC / MS / MS to quantify. A calibration curve was prepared with the peak area ratio of risedronate to the peak area of the internal standard obtained here.

Treatment of Plasma Samples

Plasma samples collected at each hour from the subject, stored at -20 ° C, were left at room temperature to be dissolved, shaken for 30 seconds, and then the plasma was collected and transferred to the test tube. It was then injected into the LC / MS / MS system (API-4000QTRAP, AB / MDX SCIEX).

Calculation of concentration in plasma

From the obtained chromatogram, the peak area ratio of risedronate to the peak area of the internal standard was determined, and the concentration of risedronate in plasma was determined from a calibration curve prepared in advance.

Example 2 Validation of Analysis Conditions

Linearity

Plasma samples spiked with standard substances (risedronate concentrations of 0.2, 0.5, 1, 2, 5, 10, 16, 20 ng / ml) and sample samples were analyzed by LC / MS / MS. Table 2 shows the calibration curve for risedronate obtained from the plasma samples. This calibration curve was used to confirm the suitability of specimen analysis and QC samples (0.5, 2, 16 ng / ml). The calibration curve equation was Y = 0.174x + 0.00413 (r = 0.9981) and showed good linearity in the range of 0.2-20 ng / ml (Fig. 3).

Table 2: Calculation of calibration curves for risedronate in plasma

Analyte Correlation coefficient (r) Calibration curve equation Risedronate 0.9981 Y = 0.0174x-0.00413

<Table 3> Accuracy of QC samples used for sample analysis

Nominal.
Conc (ng / mL) Sample Name Risedronate I.S. Ratio Measured.
Conc. (ng / mL) R.E (%) 0.5 QCA 1 11584 126239 0.0918 0.50 0.5 QCA 2 9590 118416 0.0810 0.44 -11.8 2 QCB 1 31965 98743 0.3237 1.83 -8.4 QCB 2 33209 110231 0.3013 1.70 -14.8 16 QCC 1 296070 104569 2.8313 16.21 1.3 QCC 2 195519 63376 3.0851 17.67 10.4

Trend of risedronate concentration in plasma over time of each subject

The plasma concentrations of the drugs obtained after administration of the reference drug to each subject are shown in Table 4, respectively. It was. In addition, the plasma drug concentration-time curves in the respective subjects are shown in FIGS. 5 to 8, respectively.

Table 4 Lysedronate concentration in plasma according to the time of each subject after administration of one tablet of 'Actone tablet 35 mg' (ng / ml)

Subject 0 hr 0.5 hr 1 hr 2 hr 3 hr 4 hr 6 hr 8 hr 12 hr 24 hr 48 hr A1 N.D. 3.38 8.05 11.09 8.88 5.58 3.40 1.94 0.65 0.35 N.D. A2 N.D. 5.75 7.90 6.35 6.57 4.54 2.01 0.81 0.33 N.D. N.D. B1 N.D. 10.47 16.40 11.47 8.45 7.30 3.05 1.51 0.59 0.29 N.D. B2 N.D. 5.33 7.44 5.17 3.81 2.58 1.00 0.55 0.28 N.D. N.D. Mean N.C. 6.23 9.95 8.52 6.93 5.00 2.37 1.20 0.46 0.32 N.C. SD N.C. 3.01 4.31 3.23 2.31 1.97 1.08 0.64 0.18 0.04 N.C.

* N.C. : Not calculated

* N.D. : Not detected

* Concentrations below LLOQ (0.2 ng / ml) are expressed as N.D.

Bioavailability Parameter  Calculation

The AUC value of each subject was determined using the area under the blood (plasma) concentration using 'BA Calc 2002 (Ver 1.1.1)' from the plasma drug concentration-time curves from each subject's plasma to the final blood collection time after drug administration. versus time curve), C _max (the peak plasma concentration of drug), T _max And T _{1 / 2β} were obtained. C _max was used to read the highest plasma concentration from the risedronate concentration-time curve in each subject's plasma, and T _max was used to reach the highest plasma drug concentration from the plasma drug concentration-time curve in each subject. The value read from time was used. T _{1 / 2β} is a parameter indicating whether the risedronate as the plasma elimination half-life being how quickly disappeared from the plasma. In each subject, the bioavailability parameters are summarized in <Table 5>.

Table 5 PK parameters of each subject

Subject AUC _last AUC _inf AUC% C _max T _max T _{1/2 β} A1 55.99 58.18 96.24 11.09 2.00 4.35 A2 35.64 36.61 97.35 7.90 1.00 2.04 B1 65.50 67.10 97.62 16.40 1.00 3.83 B2 25.31 26.20 96.60 7.44 1.00 2.21 Mean 45.61 47.02 96.95 10.71 1.25 3.11 SD 18.39 18.88 0.64 4.13 0.50 1.16

AUC _last : Area under the curve (AUC) to the last time point (nghr / mL)

AUC _inf : Area under the curve (AUC) to infinity (nghr / mL)

C _max: Peak plasma concentration (ng / mL)

T _max : Time to peak plasma concentration (hr)

_{T 1 / 2β: Elimination half life} in β-phase (hr)

AUC%: AUC _last / AUC _inf × 100

N.C .: Not Calculated

<References>

1. Sietsema WK et al. (1989) Antiresorptive dose-response relationships across three generations of bisphosphonates. Drugs Exp. Clin. Res . 15: 389-396.

2. Kanakis I et al. (2005) Curr. Pharmaceut. Anal . 1: 225

Goldring SR (2004) Arthritis Rheumatism 50, 2044.

4. Green JR et al. (2004) Oncologist 9: 3.

5. Fernandes C. et al. (2005) Quimica Nova 28: 852

6. Samdancioglu S. et al. (2003) J. Pharm. Sci. 28: 183

7. Ostovic D. et al. (1993) Pharmaceut. Res . 10: 470

8. Leis HJ (2004) Rapid Commun. Mass Spectrum 18: 2781

9. Sakiyama N. (2005) Biomed. Chromatogr . 9: 243

10. Mitchell DY et al. (1998) Pharmaceuti. Res . 15: 228

11. Zhu LS et al. (2006) A general approach for the quantitative analysis of bisphosphonates in human serum and urine by high-performance liquid chromatography / tandem mass spectrometry. Rapid Commun . Mass Spectrum . 20: 3421-3426.

Figure 1 schematically shows the common structure of bisphosphonate.

Figure 2 shows the mechanism of the reaction to derivatize bisphosphonate with diazomethane.

Figure 3 shows the linearity of risedronate in plasma.

FIG. 4A shows chromatograms of risedronate and internal standards before and after dosing of Subject A1.

FIG. 4b shows chromatograms of risedronate and internal standards before and after the administration of Subject A1.

Figure 5 shows the risedronate concentration in the plasma of subject A1.

FIG. 6 shows risedronate concentration in plasma of subject A2.

Figure 7 shows the risedronate concentration in the plasma of subject B1.

FIG. 8 shows risedronate concentration in plasma of test subject B2.

Figure 9 shows the risedronate concentration in the average plasma of the subject.

 (n = 4, mean ± SD)

Claims (5)

(1) adding trimethylsilyldiazomethane to derivatize bisphosphonate compounds; (2) after the derivatization process, to obtain a bisphosphonate compound derivative by removing the unreacted material and the bisphosphonate compound through solid column extraction; (3) A method of measuring the bisphosphonate compound derivative by injecting the bisphosphonate compound derivative obtained in step (2) into a liquid chromatography-mass spectrometry. The bisphosphonate compound of claim 1, wherein the bisphosphonate compound is etidronate, clodronate, tiludronate, pamidronate, neridronate, olpadronate. (olpadronate), alendronate (alendronate), ibandronate, risedronate (risedronate) and a method of measuring a bisphosphonate compound derivative, characterized in that at least one selected from the group consisting of (zoledronate). The bisphosphonate compound derivative according to claim 1, wherein trimethyloxyaryl diazomethane is used alone or in combination with at least one selected from methanol, benzene and hexane. How to measure. The method of measuring a bisphosphonate compound derivative according to claim 1, wherein the solid column is a SAX solid column. The method of claim 1, wherein the bisphosphonate compound derivative is measured using a zoleronate as an internal standard when the bisphosphonate compound derivative is measured by a liquid chromatography mass spectrometer.

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