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Method for determining plasma concentration of recombinant human parathyroid hormone (rhPTH) for injection, and metabolin thereof

A technology of parathyroid hormone and metabolites, applied in the field of determination of PTH plasma concentration, can solve the problems of expensive use, poor repeatability and stability of measurement results, etc., and achieve the effect of improving repeatability and stability

Inactive Publication Date: 2014-01-08
JIANGSU INST OF NUCLEAR MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The HPLC-MS method requires special detectors and is expensive to use; the ELISA and RIA analysis methods are simple and easy to operate, but due to the complexity of plasma components, the repeatability and stability of the measurement results are poor.

Method used

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  • Method for determining plasma concentration of recombinant human parathyroid hormone (rhPTH) for injection, and metabolin thereof
  • Method for determining plasma concentration of recombinant human parathyroid hormone (rhPTH) for injection, and metabolin thereof
  • Method for determining plasma concentration of recombinant human parathyroid hormone (rhPTH) for injection, and metabolin thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1 linear range

[0033] rhPTH(1-84) linear range:

[0034] Take the concentration as 0, 24, 55, 177, 520 pg·mL -1 rhPTH(1-84) standard solution; operate according to the ELSA-PTH operating procedure of the thyroxine radioimmunoassay kit, and the obtained standard curve is shown in figure 1 . Using linear regression at 24-520 pg·mL -1 The linear relationship within the range is good, the linear regression equation: Y=19.791X(R 2 =0.9989, figure 1 b). The multiple linear relationships of different batches were investigated respectively. PC and QC were accompanied by the standard curve to investigate the measurement accuracy (see Table 1). The relative errors of the measured values ​​of each quality control standard were less than 10%.

[0035] Take rhPTH(1-34) standard concentration as 10, 20, 40, 80, 160, 320, 640, 1280 pg·mL -1The standard solution of the thyroxine radioimmunoassay kit RK-055-08 (PTH 1-34) was operated according to the radioimmunoassa...

Embodiment 2

[0037] Embodiment 2, precision and accuracy

[0038] Take QC samples, operate according to the radio-immune operation procedure, repeat the measurement of each quality control concentration 5 times, and measure 3 consecutive analysis batches for 3 days, calculate the intra-day and inter-day precision and accuracy (Table 1), and the precision is measured by relative The standard deviation (RSD%) is expressed, and the accuracy is expressed by the relative error (RE%).

[0039] Table 1 Precision and accuracy in methodological validation (n=5)

[0040]

Embodiment 3

[0041] Embodiment 3, measure the minimum quantitative limit

[0042] Precisely prepare plasma samples containing rhPTH(1-84) and rhPTH(1-34) and dilute to 10 pg·mL -1 , operate according to the radioimmunoassay operation flow, repeat the measurement 5 times, and calculate the precision and precision. At this concentration, the average concentration of rhPTH(1-84) was (8.70±0.57) pg·mL -1 , the relative error RE is 13.0%, the relative standard deviation RSD is 6.54%; the average concentration of rhPTH(1-34) is (8.82±0.31) pg·mL -1 , the relative error RE was 11.9%, and the relative standard deviation RSD was 3.51%.

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Abstract

The invention discloses a method for determining plasma concentration of recombinant human parathyroid hormone (rhPTH) (1-84) for injection, and metabolin rhPTH (1-34) thereof, and belongs to the technical field of determination of plasma concentration of PTH. According to the method, solid phase extraction is combined with radioimmunoassay (RIA), and a plasma sample is extracted by a solid phase extraction column StyreScreen H2P, and rhPTH (1-84) is determined by thyroxine radioimmunoassay kit enzyme-linked immunosorbent assay (ELSA)-PTH; and the method comprises the following steps of: adding an rhPTH (1-84) standard substance or a treated plasma sample into ELSA TUBES, adding 125I-PTH, testing by using a compound tube, and measuring count per minute (CPM) by a gamma-counter; drawing by taking CPM value as a Y coordinate and rhPTH (1-84) concentration as an X coordinate to obtain a regression equation Y=19.791X; determining the rhPTH (1-34) by using a thyroxine radioimmunoassay kit RK-055-08 (PTH1-34); adding samples and antibodies into double compound tubes; incubating; after 125I-PTH is added into each tube, uniformly oscillating and incubating; adding goat anti-rabbit and normal rabbit serum and incubating; and measuring count B by using the gamma-counter, and drawing according to a formula B0=TB-NSB by taking B / B0 as the Y coordinate and rhPTH (1-34) concentration logarithm as the X coordinate to obtain a functional equation. By the method, the repeatability and stability of the RIA detection are improved, and reliability of determination data is improved.

Description

technical field [0001] The invention relates to a method for measuring plasma concentration of recombinant human parathyroid hormone rhPTH (1-84) and its metabolite rhPTH (1-34) for injection, belonging to the technical field of measuring methods for PTH plasma concentration. Background technique [0002] PTH is a natural parathyroid hormone secreted by the parathyroid glands. The physiologically active one is a polypeptide hormone containing 84 amino acid residues, which can stimulate new bone formation and maintain normal calcium balance and phosphorus metabolism. Recombinant human parathyroid hormone (1-84) for injection (recombinant human parathyroid hormone (1-84) for injection, abbreviated as rhPTH(1-84)) is a recombinant human parathyroid hormone containing the full-length sequence of PTH , developed by NPS-Allelix of the United States, was approved by the FDA in 2006 for the treatment of postmenopausal osteoporosis in women. rhPTH(1-84) can be partially degraded int...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/78G01N33/531
Inventor 周杏琴蔡刚明钦晓峰邹美芬徐希杰张建康曹国宪
Owner JIANGSU INST OF NUCLEAR MEDICINE