Development of rapid, simple and novel method for the validation of Abaloparatide and Teriparatide in Combined Dosage Form By RP-HPLC Method

 

Ganesh Akula1*, D. Vani2, M. Venkata Ramana3, Salla Pujitha4, Yerolla Soundarya5

1Associate Professor, Department of Chemistry,

Surabhi Dayakar Rao College of Pharmacy, JNTUH, Telangana, India.

2Student, Department of Pharmaceutical analysis,

Surabhi Dayakar Rao College of Pharmacy, JNTUH, Telangana, India.

3Principal, Department of Chemistry, Surabhi Dayakar Rao College of Pharmacy, JNTUH, Telangana, India.

4Assistant professor, Department of Pharmacology,

Surabhi Dayakar Rao College of Pharmacy, JNTUH, Telangana, India.

5Assistant Professor, Department of Pharmaceutical Analysis,

Surabhi Dayakar Rao College of Pharmacy, JNTUH, Telangana, India.

*Corresponding Author E-mail: akulaganesh@gmail.com

 

ABSTRACT:

Abaloparatide and teriparatide are prescription, self-injected, osteoanabolic (bone-building) drugs used to treat high-risk osteoporosis by strengthening bone mineral density and reducing fractures. The main aim of the present study was to develop a method to determine a Simple, Rapid, Selective, Precise, Accurate and reproducible RP-HPLC method for Simultaneous estimation of Abaloparatide and Teriparatide in bulk and pharmaceutical formulations as per ICH guidelines. The separation of the drug was achieved on Phenomenex Luna C18 (4.6×250mm, 5µm) column. The mobile phase was Acetonitrile: Phosphate Buffer (pH-4.6) in the ratio of 45:55v/v at a flow rate of 1.0mL/min elutes was monitored at 245nm, with a retention time of 2.102 and 3.537 minutes for Abaloparatide and Teriparatide respectively. The method was validated and the response was found to be linear in the drug concentration range of 6µg/mL-14µg/mL for Abaloparatide and 18µg/ml to 42µg/mL for Teriparatide and values of the slope and the correlation coefficient were found to be 77824 and 0.999 for Abaloparatide and 10515 and 0.999 for Teriparatide respectively. The LOD and LOQ values of Abaloparatide and Teriparatide were found to be 0.6µg/mL and1.8µg/mL and 0.8µg/mL and 2.4µg/mL respectively. The method was also employed on formulation and the recovery of Abaloparatide and Teriparatide were found to be 100.351 and 100.93 respectively.

 

KEYWORDS: Abaloparatide and Teriparatide, RP-HPLC, Accuracy, Precision, ICH Guidelines.

 

 

1. INTRODUCTION:

Different analytical methods have been reported for the estimation and validation of abaloparatide and teriparatide.1 According to WHO guidelines, validation is defined as a documented act that demonstrates or verifies that any part, substance, method, machine, system, or activity and it confirming that all GMPs have been adhered to successfully throughout the manufacturing process and that the anticipated outcomes will materialise.2  RP-HPLC method having simple composition of  mobile phase. Hence an attempt has been made to develop and validate a novel, simple and sensitive cost-effective rapid RP-HPLC method in validated according to international conference on harmonization (ICH) guidelines guidelines.3,4,5 The principle behind RP-HPLC is hydrophobic interaction.6  The method for the estimation of abaloparatide and teriparatide in their combined dosage form and to validate the method with respect to specificity, accuracy, precision, linearity and range.7,8,9,10

 

Abaloparatide is an analog of PTHrP (parathyroid hormone-related protein) with the molecular formula C174H300N56O49 and Molecular weight:3961.0 Da11. It was approved in April 28, 2017 by the FDA (as Tymlos) for the treatment of postmenopausal women with osteoporosis at high risk for fracture12. It have potential to widen the anabolic window for bone therapeutics, stimulating bone formation with a limited effect on bone resorption and mineral mobilization. acts as an agonist on PTH type 1 receptor (PTH1R) and activates both G protein–mediated cAMP signaling and β-arrestin-mediated ERK-1/2 signaling also binds to binds to RG conformation of PTH1R that results in more transient cell signalling responses.13

 

Teriparatide (recombinant human parathyroid hormone/PTH) with Molecular Formula: C181H291N55O51S2 Molecular weight:4117.715 Da with a potent anabolic agent14 used in the treatment of osteoporosis.  Endogenous PTH is the primary regulator of calcium and phosphate metabolism in bone and kidney and stimulates new bone formation leading to increased bone mineral density that helps to decrease the risk of getting a fracture.15

 

2. MATERIALS AND METHODS:

Optimized Chromatographic Conditions:

Instrument used:

Waters HPLC with auto sampler and PDA Detector 996 model.

Temperature:

35ºC

Column:

Phenomenex Luna C18 (4.6×250mm, 5µm) particle size

Buffer:

Potassium dihydrogen Phosphate

pH:

4.6

Mobile phase:

Acetonitrile: Phosphate Buffer (45:55 v/v)

Flow rate:

1ml/min

Wavelength:

245 nm

Injection volume:

10 ml

Run time:

7 min

 

Preparation of Potassium dihydrogen Phosphate (KH2PO4) buffer (pH-4.6):

Dissolved 6.8043 of potassium dihydrogen phosphate in 1000ml HPLC water and adjusted the pH 4.6 with diluted orthophosphoric acid. Filtered and sonicated the solution by vacuum filtration and ultrasonication.

 

Preparation of Mobile Phase:

Accurately measured 450ml (45%) of Methanol, 550ml of Phosphate buffer (55%) were mixed and degassed in digital ultra sonicater for 15 minutes and then filtered through 0.45µ filter under vacuum filtration.

The Mobile phase was used as the diluent.

 

Preparation of Standard Solutions of Abaloparatide and Teriparatide:

Accurately weighed and transferred 10mg of Abaloparatide and 10mg of Teriparatide working standard into a 10ml of clean dry volumetric flasks added 7mL of Diluents and sonicated to dissolve it completely and made volume up to the mark with the same solvent. (Stock solution) Further pipette 0.1ml of the above Abaloparatide and 0.3ml of the Teriparatide stock solutions into a 10ml volumetric flask and dilute up to the mark with Diluent.

 

VALIDATION PARAMETERS:

Drug Specificity:

Preparation of Sample Solution:

An average weight of Tablet and crushed in a mortor by using pestle and weight 10mg weight of Abaloparatide and Teriparatide sample into a 10mL clean dry volumetric flask and added 7mL of Diluent and sonicated to dissolve it completely and made volume up to the mark with the same solvent.  Further pipette 0.1ml of the above Abaloparatide and 0.3ml of the Teriparatide stock solutions into a 10ml volumetric flask and diluted up to the mark with Diluent.

 

Preparation of drug solution for Linearity (Level I-V):

Pipette out 0.06ml and 0.18ml,0.08ml and 0.24ml, 0.1 ml and 0.3ml, 0.12 ml and 0.36ml, 0.14ml and 0.42ml of stock solutions to prepare 6 ppm and 18ppm, 8ppmand 24ppm, 10ppm and 30ppm, 12ppm and 36ppm, 14ppm and 42ppm of Abaloparatide and Teriparatide solutions respectively.

 

Injected each level into the chromatographic system and measured the peak area.

 

Precision:

Repeatability:

The standard solutions were injected for five times measured the area and %RSD for the area of five replicate injections was calculated.

 

Intermediate Precision:

Intermediate precision /Ruggedness was performed for 2 days by injecting 6 replicated injections by maintaining same conditions.

 

Accuracy:

Preparation of 50%, 100% and 150% Standard Stock Solutions:

Pipette 0.05ml and 0.15ml, 0.1ml and 0.3ml, 0.15ml and 0.45ml of Abaloparatide and Teriparatide stock solutions respectively into a 10ml volumetric flask and diluted up to the mark with Diluent. Inject the Three replicate injections of individual concentrations. Recorded the chromatograms and measured the peak responses.

 

Robustness:

The sample was analyzed by variation in flow conditions and mobile phase. Flow conditions at 0.9 ml/min and 1.1ml/min instead of 1ml/min and Acetonitrile: Phosphate Buffer was taken in the ratio and 50:50, 40:60 instead (45:55), remaining conditions are same. 10µl of the above sample was injected and chromatograms were recorded.

 

3. RESULTS

 

Figure 1: Chromatogram showing blank (mobile phase preparation)

 

Table 1: Results of system suitability

S. No

Name

Rt

Area

Height

USP plate count

USP Tailing

USP Resolution

 

1

Abaloparatide

2.117

765843

69587

5589

1.9

-

Mean 765855.6

2

Abaloparatide

2.118

766594

69854

5576

1.6

-

3

Abaloparatide

2.116

765487

70211

5658

1.6

-

Std. Dev 466.6522

4

Abaloparatide

2.109

765928

69213

5642

1.7

-

5

Abaloparatide

2.102

765426

69558

5685

1.6

-

% RSD 0.060932

6

Teriparatide

3.547

2534658

190058

5365

1.2

2.07

Mean 2535034

7

Teriparatide

3.539

2536854

190052

5348

1.4

2.05

8

Teriparatide

3.547

2535879

190078

5389

1.5

2.0

Std. Dev 1183.309

9

Teriparatide

3.565

2533564

190035

5347

1.6

2.01

10

Teriparatide

3.537

2534214

190085

5364

1.6

2.01

% RSD0.046678

 

Table-:2 Peak results for assay standard

S. No

Name

Rt

Area

Height

USP Resolution

USP Tailing

USP plate count

Injection

1

Abaloparatide

2.102

759868

71255

 

1.7

5689

1

2

Teriparatide

3.537

2458754

215654

2.04

1.6

5362

1

3

Abaloparatide

2.105

759458

72541

 

1.7

5748

2

4

Teriparatide

3.552

2465885

226565

2.00

1.6

5452

2

5

Abaloparatide

2.112

759245

72584

 

1.7

5584

3

6

Teriparatide

3.560

2489578

221542

2.04

1.6

5456

3

 

Table 3: Peak results for Assay sample

S.No

Name

Rt

Area

Height

USP Resolution

USP Tailing

USP plate count

Injection

1

Abaloparatide

2.120

756985

68958

 

0.98

7253

1

2

Teriparatide

3.536

2569856

198564

2.06

1.23

8836

1

3

Abaloparatide

2.120

758745

69857

 

1.05

6530

2

4

Teriparatide

3.537

2598654

195682

2.04

0.99

7270

2

5

Abaloparatide

2.102

756848

69588

 

1.7

7586

3

6

Teriparatide

3.537

2587454

192541

2.04

1.6

8371

3

 

Table 4: Linearity Chromatographic Data

Drug

Concentration

mg/ml

Average Peak Area

Drug

Concentration

mg/ml

Average Peak Area

Abaloparatide

 

6

467849

Teriparatide

 

18

1789546

8

619854

24

2456987

10

768784

30

3085985

12

928977

36

3759864

14

1095698

42

4406589

18

1789546

Figure 2: Calibration Graph for Abaloparatide 

 

Figure 3: Calibration Graph for Teriparatide

 

Linearity Plot:

The plot of Concentration (x) versus the Average Peak Area (y) data of Teriparatide is a straight line.

Slope (m) = 10515 Intercept (c) = 45591   Correlation Coefficient (r) =   0.999

 

Precision:

Table 5: Results of method precision

S. No

Name

Injections

Rt

Area

Height

USP plate count

USP Tailing

 

1

Abaloparatide

Inj-1

2.108

766854

702564

5685

1.6

Mean

766682

2

Inj-2

2.105

765884

698789

5584

1.4

3

Inj-3

2.113

765842

701235

5521

1.6

Std. Dev

1357.973

4

Inj-4

2.109

768985

700124

5525

1.9

5

Inj-5

2.109

765845

698986

5578

1.7

% RSD 0.177123

6

Teriparatide

Inj-1

3.552

2569865

2231111

5365

1.6

Mean

2570013

7

Inj-2

3.550

2578474

2674210

5425

1.6

8

Inj-3

3.564

2568985

2231261

5368

1.5

Std. Dev

15309.45

9

Inj-4

3.564

2586845

2421301

5359

1.5

10

Inj-5

3.565

2545898

2324710

5498

1.6

% RSD

0.595695

 

Table 6: Results of Intermediate precision on Day 1 and Day 2 for Abaloparatide

S. No.

Day

Name

Rt

Area

Height

USP plate count

USP Tailing

 

1

 

 

Day-1

Abaloparatide Inj-1

2.108

758955

68986

5785

1.6

Mean

758590.3

2

Abaloparatide Inj-2

2.105

759869

68957

5698

1.4

3

Abaloparatide Inj-3

2.113

758985

68545

5689

1.6

Std. Dev

1339.793

4

Abaloparatide Inj-4

2.109

756894

68952

5781

1.9

5

Abaloparatide Inj-5

2.109

759854

68595

5785

1.7

% RSD

0.176616

6

Abaloparatide Inj-6

2.102

756985

68952

5693

1.6

7

 

Day-2

Abaloparatide Inj-1

2.102

766895

69858

5586

1.5

Mean

766128.5

8

Abaloparatide Inj-2

2.105

765988

69854

5636

1.6

9

Abaloparatide Inj-3

2.112

766532

69824

5432

1.6

Std. Dev

567.7234

10

Abaloparatide Inj-4

2.113

766214

69875

5468

1.6

11

Abaloparatide Inj-5

2.109

765897

69854

5546

1.9

% RSD

0.074103

12

Abaloparatide Inj-6

2.109

765245

69848

5507

1.7

 

Table 7: Results of Intermediate precision on Day 1 and Day 2 for Teriparatide

S. No

Day

 

Name

Rt

Area

Height

USP plate count

USP Tailing

USP Resolution

 

1

 

 

Day-1

Teriparatide Inj-1

3.552

2659852

190025

5485

1.5

2.04

Mean

2655079

2

Teriparatide Inj-2

3.550

2648574

190048

5421

1.6

2.03

3

Teriparatide Inj-3

3.564

2659865

190054

5468

1.6

2.01

Std. Dev

5242.086

4

Teriparatide Inj-4

3.564

2658547

190078

5487

1.6

2.05

5

Teriparatide Inj-5

3.565

2648981

190016

5492

1.6

2.02

% RSD

0.197436

6

Teriparatide Inj-6

3.537

2654652

190057

5463

1.6

2.03

7

 

 

Day-2

Teriparatide Inj-1

3.537

2653254

190110

5428

1.6

7.98

Mean

2653678

8

Teriparatide Inj-2

3.552

2648985

190058

5452

1.6

6.4

9

Teriparatide Inj-3

3.560

2658213

190142

5498

1.6

8.9

Std. Dev

4313.355

10

Teriparatide Inj-4

3.564

2653652

190031

5442

1.5

8.3

11

Teriparatide Inj-5

3.564

2648978

190058

5489

1.5

7.5

% RSD

0.162543

12

Teriparatide Inj-6

3.565

2658985

190047

5463

1.6

5.3

 

Table 8: The accuracy results

%Concentration

(at specification Level)

 

Drug

Area

Amount Added (ppm)

Amount Found (ppm)

% Recovery

Mean Recovery

50%

 

Abaloparatide

392891.7

5

5.027

100.540%

100.351%

100%

781996

10

10.026

100.260%

150%

1171988

15

15.038

100.253%

50%

 

Teriparatide

204962

15

15.156

101.040%

100.93%

100%

365018

30

30.378

101.260%

150%

521064.3

45

45.218

100.484%

 

Table 9: Result for Robustness

Drug

Parameter used for sample analysis

Peak Area

Retention Time

Theoretical plates

Tailing factor

 

Abaloparatide

Actual Flow rate of 1.0mL/min

765789

2.102

5587

1.7

Less Flow rate of 0.9mL/min

758698

2.330

5458

1.7

More Flow rate of 1.1mL/min

7689584

1.950

5696

1.7

Less organic phase

758412

2.290

5586

1.4

More organic phase

769852

1.998

5355

1.5

 

Teriparatide

Actual Flow rate of 1.0mL/min

2532158

3.537

5398

1.6

Less Flow rate of 0.9mL/min

2458692

3.885

5329

1.7

More Flow rate of 1.1mL/min

2658642

3.263

5256

1.7

Less organic phase

2452148

4.435

5214

1.2

More organic phase

2653894

3.009

5524

1.0

 

Limit Of Detection:

Abaloparatide:0.6µg/ml

Teriparatide: 0.8µg/ml

Limit of Quantitation:

Abaloparatide: 1.8µg/ml

Teriparatide:2.4µg/ml

 

4. CONCLUSION:

The proposed method was found to be novel, simple, precise, accurate, rapid and specific for determination of Abaloparatide and Teriparatide in combined dosage form. Preparation of mobile phase was simple and economical. The method was validated for precision, accuracy and linearity. The RSD values for all the parameters were found to be less than 2, which indicates the method is fair agreement for obtained results. Hence this method can be easily and conveniently intended for the routine analysis of Abaloparatide and Teriparatide in combined dosage form.

 

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11.   https://pubchem.ncbi.nlm.nih.gov/compound/Abaloparatide

12.   https://en.wikipedia.org/wiki/Abaloparatide

13.   https://www.drugbank.ca/drugs/DB06285

14.   https://pubchem.ncbi.nlm.nih.gov/compound/Teriparatide

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Received on 02.03.2026      Revised on 09.04.2026

Accepted on 11.05.2026      Published on 27.05.2026

Available online from May 30, 2026

Asian J. Research Chem.2026; 19(3):177-181.

DOI: 10.52711/0974-4150.2026.00029

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