Development and Validation of a LC-MS/MS Assay for Quantification of Parathyroid Hormone (PTH 1-34) in human Plasma

Sulaiman Al Riyami; Jonathan C Y Tang; John Dutton; Christopher Washbourne; Hillel Galitzer; William Fraser

Development and Validation of a LC-MS/MS Assay for Quantification of Parathyroid Hormone (PTH 1-34) in human Plasma

Sulaiman Al Riyami (Lead Author), Jonathan C Y Tang, John Dutton, Christopher Washbourne, Hillel Galitzer, William Fraser

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Abstract

Background: Teriparatide [recombinant human PTH (1-34)] is an osteoanabolic agent for treatment of osteoporosis. The effect on bone decreases the risk of vertebral and non-vertebral fractures and increases bone mineral density (BMD) in post-menopausal women with osteoporosis. Measurement of PTH (1-34) is valuable in assessing treatment response and concordance with therapy.Aim: To develop and validate a method for quantification of PTH (1-34) using Liquid chromatography tandem mass spectrometry (LC-MS/MS) and to perform comparison with a commercial immunoassay.
Method: Sample extraction was developed using a Waters (Milford, MA, USA) Oasis® HLB µElution solid phase extraction. Quantification m/z transition 589>656 was used on Waters/Micromass® Quattro Ultima™ Pt mass spectrometer to measure PTH (1-34) in human plasma using rat PTH (1-34) as internal standard. Validation criteria were carried out against industry standards. PTH (1-34) results obtained from human subjects given Teriparatide (Fortsteo, Eli Lilly, IN, USA) (n=390) were compared against results obtained from an immunoassay (IDS; Boldon Tyne and Wear. UK).
Results and Discussion: LC-MS/MS produced a linear calibration curve from 10 to 2000 pg/mL (r2 >0.990). The LLoQ and LLoD for PTH (1-34) were 10 pg/mL and 2.1 pg/mL respectively. The inter- /intra-assay precision (CV%) of the method were <9.8 and <7.8% and accuracy of >98.3% for four QCs (20, 100, 200, and 800 pg/mL). The mean recovery of PTH (1-34) was 107.2%. Method comparison between the LC-MS/MS and immunoassay using human EDTA plasma samples showed a high correlation (r2 = 0.950). A concentration-dependent, negative bias of 35.5% was observed across the range of 0 – 800 pg/mL. The immunoassay showed a 7% cross reactivity to human PTH (1-84) and 44% to rat PTH (1-34), no interference was observed in the LC-MS/MS method. Matrix effect and cross reactivity to human PTH (1-84) in the immunoassay were the likely contributing factors to the bias between the methods. The oxidised form of PTH (1-34) does not interfere with our LC-MS/MS method.
Conclusion: Our LC-MS/MS method demonstrated linearity over the calibration range, good precision and accuracy, excellent analyte recovery, and negligible matrix effects. The method was successfully used for measurements of PTH (1-34) in rat and human plasma.

Original language	English
Publication status	Published - 10 Sep 2017
Event	ASBMR 2017 Annual Meeting - Colorado Convention Center, ASBMR Discovery Hall, United States Duration: 8 Sep 2017 → 11 Sep 2017

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Conference	ASBMR 2017 Annual Meeting
Country/Territory	United States
Period	8/09/17 → 11/09/17

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PosterFinal published version, 1.58 MBLicence: Unspecified

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@conference{65f5030fa04f4707b05d74f643f13902,

title = "Development and Validation of a LC-MS/MS Assay for Quantification of Parathyroid Hormone (PTH 1-34) in human Plasma",

abstract = "Background: Teriparatide [recombinant human PTH (1-34)] is an osteoanabolic agent for treatment of osteoporosis. The effect on bone decreases the risk of vertebral and non-vertebral fractures and increases bone mineral density (BMD) in post-menopausal women with osteoporosis. Measurement of PTH (1-34) is valuable in assessing treatment response and concordance with therapy.Aim: To develop and validate a method for quantification of PTH (1-34) using Liquid chromatography tandem mass spectrometry (LC-MS/MS) and to perform comparison with a commercial immunoassay. Method: Sample extraction was developed using a Waters (Milford, MA, USA) Oasis{\textregistered} HLB µElution solid phase extraction. Quantification m/z transition 589>656 was used on Waters/Micromass{\textregistered} Quattro Ultima{\texttrademark} Pt mass spectrometer to measure PTH (1-34) in human plasma using rat PTH (1-34) as internal standard. Validation criteria were carried out against industry standards. PTH (1-34) results obtained from human subjects given Teriparatide (Fortsteo, Eli Lilly, IN, USA) (n=390) were compared against results obtained from an immunoassay (IDS; Boldon Tyne and Wear. UK). Results and Discussion: LC-MS/MS produced a linear calibration curve from 10 to 2000 pg/mL (r2 >0.990). The LLoQ and LLoD for PTH (1-34) were 10 pg/mL and 2.1 pg/mL respectively. The inter- /intra-assay precision (CV%) of the method were <9.8 and <7.8% and accuracy of >98.3% for four QCs (20, 100, 200, and 800 pg/mL). The mean recovery of PTH (1-34) was 107.2%. Method comparison between the LC-MS/MS and immunoassay using human EDTA plasma samples showed a high correlation (r2 = 0.950). A concentration-dependent, negative bias of 35.5% was observed across the range of 0 – 800 pg/mL. The immunoassay showed a 7% cross reactivity to human PTH (1-84) and 44% to rat PTH (1-34), no interference was observed in the LC-MS/MS method. Matrix effect and cross reactivity to human PTH (1-84) in the immunoassay were the likely contributing factors to the bias between the methods. The oxidised form of PTH (1-34) does not interfere with our LC-MS/MS method. Conclusion: Our LC-MS/MS method demonstrated linearity over the calibration range, good precision and accuracy, excellent analyte recovery, and negligible matrix effects. The method was successfully used for measurements of PTH (1-34) in rat and human plasma.",

author = "{Al Riyami}, Sulaiman and Tang, {Jonathan C Y} and John Dutton and Christopher Washbourne and Hillel Galitzer and William Fraser",

year = "2017",

month = sep,

day = "10",

language = "English",

note = "ASBMR 2017 Annual Meeting ; Conference date: 08-09-2017 Through 11-09-2017",

}

TY - CONF

T1 - Development and Validation of a LC-MS/MS Assay for Quantification of Parathyroid Hormone (PTH 1-34) in human Plasma

AU - Al Riyami, Sulaiman

AU - Tang, Jonathan C Y

AU - Dutton, John

AU - Washbourne, Christopher

AU - Galitzer, Hillel

AU - Fraser, William

PY - 2017/9/10

Y1 - 2017/9/10

N2 - Background: Teriparatide [recombinant human PTH (1-34)] is an osteoanabolic agent for treatment of osteoporosis. The effect on bone decreases the risk of vertebral and non-vertebral fractures and increases bone mineral density (BMD) in post-menopausal women with osteoporosis. Measurement of PTH (1-34) is valuable in assessing treatment response and concordance with therapy.Aim: To develop and validate a method for quantification of PTH (1-34) using Liquid chromatography tandem mass spectrometry (LC-MS/MS) and to perform comparison with a commercial immunoassay. Method: Sample extraction was developed using a Waters (Milford, MA, USA) Oasis® HLB µElution solid phase extraction. Quantification m/z transition 589>656 was used on Waters/Micromass® Quattro Ultima™ Pt mass spectrometer to measure PTH (1-34) in human plasma using rat PTH (1-34) as internal standard. Validation criteria were carried out against industry standards. PTH (1-34) results obtained from human subjects given Teriparatide (Fortsteo, Eli Lilly, IN, USA) (n=390) were compared against results obtained from an immunoassay (IDS; Boldon Tyne and Wear. UK). Results and Discussion: LC-MS/MS produced a linear calibration curve from 10 to 2000 pg/mL (r2 >0.990). The LLoQ and LLoD for PTH (1-34) were 10 pg/mL and 2.1 pg/mL respectively. The inter- /intra-assay precision (CV%) of the method were <9.8 and <7.8% and accuracy of >98.3% for four QCs (20, 100, 200, and 800 pg/mL). The mean recovery of PTH (1-34) was 107.2%. Method comparison between the LC-MS/MS and immunoassay using human EDTA plasma samples showed a high correlation (r2 = 0.950). A concentration-dependent, negative bias of 35.5% was observed across the range of 0 – 800 pg/mL. The immunoassay showed a 7% cross reactivity to human PTH (1-84) and 44% to rat PTH (1-34), no interference was observed in the LC-MS/MS method. Matrix effect and cross reactivity to human PTH (1-84) in the immunoassay were the likely contributing factors to the bias between the methods. The oxidised form of PTH (1-34) does not interfere with our LC-MS/MS method. Conclusion: Our LC-MS/MS method demonstrated linearity over the calibration range, good precision and accuracy, excellent analyte recovery, and negligible matrix effects. The method was successfully used for measurements of PTH (1-34) in rat and human plasma.

AB - Background: Teriparatide [recombinant human PTH (1-34)] is an osteoanabolic agent for treatment of osteoporosis. The effect on bone decreases the risk of vertebral and non-vertebral fractures and increases bone mineral density (BMD) in post-menopausal women with osteoporosis. Measurement of PTH (1-34) is valuable in assessing treatment response and concordance with therapy.Aim: To develop and validate a method for quantification of PTH (1-34) using Liquid chromatography tandem mass spectrometry (LC-MS/MS) and to perform comparison with a commercial immunoassay. Method: Sample extraction was developed using a Waters (Milford, MA, USA) Oasis® HLB µElution solid phase extraction. Quantification m/z transition 589>656 was used on Waters/Micromass® Quattro Ultima™ Pt mass spectrometer to measure PTH (1-34) in human plasma using rat PTH (1-34) as internal standard. Validation criteria were carried out against industry standards. PTH (1-34) results obtained from human subjects given Teriparatide (Fortsteo, Eli Lilly, IN, USA) (n=390) were compared against results obtained from an immunoassay (IDS; Boldon Tyne and Wear. UK). Results and Discussion: LC-MS/MS produced a linear calibration curve from 10 to 2000 pg/mL (r2 >0.990). The LLoQ and LLoD for PTH (1-34) were 10 pg/mL and 2.1 pg/mL respectively. The inter- /intra-assay precision (CV%) of the method were <9.8 and <7.8% and accuracy of >98.3% for four QCs (20, 100, 200, and 800 pg/mL). The mean recovery of PTH (1-34) was 107.2%. Method comparison between the LC-MS/MS and immunoassay using human EDTA plasma samples showed a high correlation (r2 = 0.950). A concentration-dependent, negative bias of 35.5% was observed across the range of 0 – 800 pg/mL. The immunoassay showed a 7% cross reactivity to human PTH (1-84) and 44% to rat PTH (1-34), no interference was observed in the LC-MS/MS method. Matrix effect and cross reactivity to human PTH (1-84) in the immunoassay were the likely contributing factors to the bias between the methods. The oxidised form of PTH (1-34) does not interfere with our LC-MS/MS method. Conclusion: Our LC-MS/MS method demonstrated linearity over the calibration range, good precision and accuracy, excellent analyte recovery, and negligible matrix effects. The method was successfully used for measurements of PTH (1-34) in rat and human plasma.

UR - http://www.asbmr.org/education/AbstractDetail?aid=e4abcd35-f883-45eb-a82e-f2da8c3e6fe4

M3 - Poster

T2 - ASBMR 2017 Annual Meeting

Y2 - 8 September 2017 through 11 September 2017

ER -

Development and Validation of a LC-MS/MS Assay for Quantification of Parathyroid Hormone (PTH 1-34) in human Plasma

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LC-MS/MS measurement of parathyroid hormone PTH (1-34): Use in studying oral PTH (1-34) administration and possible diagnostic application in pseudohypoparathyroidism.

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