A Comprehensive Review of Analytical Methods for the Determination of Semaglutide in Pharmaceutical and Biological Samples

Diabetes is one of the most common metabolic and civilization diseases. Polydipsia, dray skin, frequent urination, fatigue, blurred vision, unexplained weight loss and even depression can be symptoms of excessively high levels oof sugar in human blood stream.

Dysfunctional secretion of pancreatic hormone insulin plays a major role in this disorder. A deficiency of insulin or a lack of sensitivity of pheripheral receptors to insulin leads to hypoglycemia. Glucose is overproduced and undelutlize as an energy source. These are several types of diabetes

The most common forms include: 

1) Type-1

2) Type-2

3) Gestational diabetes

Type-1

Type -1 diabetes (also known as juvenile diabetes) occurs in children and teenagers. Initial symptoms appeal very quickly and process rapidly This form of diabetes has an autoimmune cause. Endogenous antibodies damage pancreatic beta cells that are responsible for producing insulin. An insufficient amount of this hormone in the blood stream leads to hyperglycemia. Diabetes use natural or synthetic insulin as the only method of medicinal treatment.

Type-2

Type -2 diabetes usually appears in adult. A lack of physical activity, obesity diet high in sugar content are the main contributing factors to elevated levels of sugar in blood. This type of diabetes is connected with insulin resistance. Insulin resistance occurs when human cells respond inadequately to insulin impairing their ability to efficiently absorb and store glucose from the blood stream. Treatment begins with lifestyle modifications, followed by antidiabetic drugs and insulin.

Gestational diabetics

It can develop during pregnancy between 33% to 70 % of gestational diabetes is diagnosed in early pregnancy (early GD is defined by hypoglycemia occurring before 20 weeks of gestation). Early gestational diabetes is associated with worse pregnancy outcomes compared to women diagnose with late gestational diabetes (hyperglycemia typically from 24 weeks to 28 weeks of gestation). In most circumstances the condition resolves after childbirth.

Glucagon-like peptide-1 (GLP-1) agonists are an innovative class of drugs approved to treat type 2 diabetes mellitus. Semaglutide (C187H291N45O59) is an analog that is 94% similar to human GLP-1. Two amino acids substitutions and one acylation were made as a major structural modification to avoid dipeptidyl peptidase-4 (DPP-4) cleavage and to increase its affinity of albumin .

In fact, GLP-1 cannot be used for clinical purposes as it is rapidly degraded by dipeptidyl peptidase-4 (DPP-4) enzymes. There are three important modifications in semaglutide that allow its clinical use: the substitution of the amino acid alanine with the alpha-aminoisobutyric acid at position 8, substitution of lysine with arginine at position 34, and the attachment of a linker and a C18 di-acid chain at position 26. All these modifications allow strong binding to albumin, which reduces its renal clearance and degradation by DDP-4 enzymes. Thanks to these modifications, the half-life of semaglutide is about a week, compared to the few minutes of GLP-1.

It is a drug which is available as injectable as well as oral dosage form. Once weekly subcutaneous injectable semaglutide (Ozempic) was approved by the US food and administration (FDA) in 2017 and by the European medicines Agency (EMA) in 2018 for the treatment of type 2 diabetes. Subsequently, a daily oral tablet of semaglutide (Rybelsus) was approved in 2019 by the FDA and in 2020 by EMA. In 2021, the US FDA also approved semaglutide subcutaneous injection (Wegovy) for chronic weight management in patients with obesity (or) overweigh, which was the first since 2014.

Therapeutic peptides including glucagon-like peptide-1 (GLP-1) receptor agonists are emerging as a new effective generation of therapeutic because of the advancements in modification chemistry and analytical technologies and rapid progression in proteomics. Moreover, peptide drug development has expanded with recent advances in structural biology, recombinant biologics, and new synthetic technologies. Peptide-based therapeutics has also attracted attention because of their exceptional specificity, minimal immunogenicity, low toxicity, and therapeutic efficacy. Since 2000, more than 30 non-insulin peptide drugs have been approved worldwide. Nonetheless, quantitative peptide bioanalysis remains a challenge due to their high molecular weight, complex structures, significant endogenous interference, low in vivo concentrations, and susceptibility to non-specific binding. These aspects of peptides can lead to substantial loss during preparation and increase the risk of matric interference .

MECHANISM OF ACTION

Initially intended to control blood glucose levels in patients with type 2 diabetes, semaglutide, a potent glucagon-like peptide 1 analogue, has been established as an effective weight loss treatment by controlling appetite. Integrating the latest clinical trails, semaglutide in patients with or without diabetes presents significant therapeutic efficacy in ameliorating cardiometabolic risk factors and physical functioning, independent of body weight reduction. Semaglutide may modulate adipose tissue browning, which enhances human metabolism and exhibits possible benefits in skeletal muscle degeneration, accelerated by obesity and ageing. This may be attributed to anti-inflammatory, mitochondrial biogenesis, antioxidant and autophagy-regulating effects. However, most of the supporting evidence on the mechanistic actions of semaglutide is preclinical, demonstrated in rodents and not actually confirmed in humans, therefore warranting caution in the interpretation.

PHARMACOKINETICS OF SEMAGLUTIDE

Absorption

Absolute bioavailability of semaglutide is 89%. Maximum concentration of semaglutide is reached 1-3 days post dose.

Similar exposure is achieved with subcutaneous administration of semaglutide in the abdomen, thigh, or upper arm.

In patients with type 2 diabetes, semaglutide exposure increases in a dose-proportional manner for once-weekly doses of 0.5 mg, 1 mg, and 2 mg. Steady-state exposure is achieved following 4-5 weeks of once-weekly administration. In patients with type 2 diabetes, the mean population-PK estimated steady-state concentrations following once weekly subcutaneous administration of 0.5 mg and 1 mg semaglutide were approximately 65.0 ng/ml and 123.0 ng/ml, respectively. In the trail comparing semaglutide 1mg and 2mg, the mean steady state concentrations were 111.1 ng/ml and 222.1 ng/ml, respectively.

Distribution

The mean apparent volume of distribution of semaglutide following subcutaneous administration in patients with type 2 diabetes is approximately 12.5L. Semaglutide is extensively bound to plasma albumin(>99%).

Elimination

The apparent clearance of semaglutide in patients with type 2 diabetes is approximately 0.05 L/h. With an elimination half-life of approximately 1 week, semaglutide will be present in the circulation for about 5 weeks after the last dose.

PHARMACODYNAMICS OF SEMAGLUTIDE

Semaglutide lowers fasting  and postprandial blood glucose and reduces body weight. All pharmacodynamic evaluations were performed after 12 weeks of treatment (including dose escalation) at steady state with semaglutide 1 mg.

Fasting and Postprandial Glucose

Semaglutide reduces fasting and postprandial glucose concentrations. In patients with type 2 diabetes, treatment with semaglutide 1 mg resulted in reductions in glucose in terms of absolute change from baseline and relative reduction compared to placebo of 29 mg/dL (22%) for fasting glucose, 74 mg/dL (36%) for 2-hour postprandial glucose, and 30 mg/dL (22%) for mean 24-hour glucose concentration.

Insulin Secretion

Both first-and second-phase  insulin secretion are increased in patients with type 2 diabetes treated with OZEMPIC compared with placebo.

Glucagon Secretion

Semaglutide lowers the fasting and postprandial glucagon concentrations. In patients with type 2 diabetes, treatment with semaglutide resulted in the following relative reductions in glucagon to placebo, fasting glucagon (8%), postprandial glucagon response (14-15%), and mean 24 hour glucagon concentration (12%).

ANALYTICAL METHODS FOR SEMAGLUTIDE

The analytical methods used to evaluate Semaglutide, providing an overview of different analytical methods and emphasizing their applications in pharmaceutical analysis.

1. RP-UPLC Method:

The objective of the study was to develop was to develop RP-UPLC method for the determination of purity of semaglutide in bulk and pharmaceutical dosage form.

This method was developed by SUBHA HARIKA PENMETSA and RAJA SUNDARARAJAN in the year 19-october-2018.

The chromatographic separation was achieved which acquity BEH C18 [50mm × 1.6mm] 1.8µm column thermostated at 30°c with mobile phases containing 0.01 N potassium dihydrogen phosphate [3.2 pH] : Acetonitrile in the ratio of 50:50 v/v. The flow rate was maintained at 0.4 ml/min and injection volume was found to be 0.50 µL. The detection was done at 292nm using TUV detector. The retention time was found to be 1.026 min. Linearity study was carried out between 25% to 150% concentration levels and R2 value was found to be as 0.999. The % RSD of repeatability and intermediate precision was found to be 0.5 and 0.6 µg/ml. The LOD and LOQ were found to be 0.086 µg/ml and 0.261 µg/ml, respectively. Forced degradation was carried out under various stress conditions to demonstrate the stability-indicating capability of the developed UPLC method. The proposed method was found to be simple, precise, accurate and validated according to the international conference on Harmonization guidelines.

2. RP-HPLC + Forced Degradation :

The objective is for the development of a specific and simple method to trace semaglutide presence in active pharma ingredient and pharma dosages .

This method was developed by ARUN KUMAR KUNA, GANAPATY S, RADHA GV in the year 2019-april 17.

As part of the study on semaglutide drug solvents of HPLC instrument (empower software) with PDA detector ultrasonicator (make : labman) and PH meter are used .The method was optimized with mobile phase with a composition of buffer and solvent were of 60:40 % V/V flow maintained was 1.0 ml/min, the injection volume of 10ml run time was 150×4.6 mm, 5m. Results for the developed method are accurate and specific. The detection wavelength was 292 nm, the retention time for semaglutide was 2.689 min, linearity resulted with r₂=0.9998% RSP for precision was 1.0% mean recovery for accuracy was in the range of 99.73 to 100.29, This study report is for industrial application of determining semaglutide presence in pharma ingredient and dosages.

3. Stability – Indicating RP-HPLC :

A new specific, economic and selective, accurate, precise and robustness Reverse Phase High Performance Liquid Chromatography was developed for the quantification of semaglutide in pharmaceutical substances and product.

This method was developed by MERUGU MANASA , VIJEY AANANDHI M in the year 2021.

Chromatographic separation was achieved by C18  column [Azilent C18 150×4.6mm] is used as stationary phase and 0.01 N potassium dihydrogen ortho phosphate: acetonitrile [50:50] used as a mobile phase at a flow rate of 0.1 ml/min and monitored at 230nm. The run time was 5min. The retention time of semaglutide was found to be 2.222 min. To fulfill international conference on Harmonisation requirements developed method was validated. Validation parameters include system suitability, specificity, linearity, accuracy, precision, intermediate precision and robustness. Calibration curve was linear over the concentration range of 7.42 µg/ml from regression analysis relative correlation coefficient (R2) value was found to be 0.999. Accuracy and precision results were within the limits. LOD and LOQ were found to be 0.007 µg/ml and 0.022 µg/ml respectively. Semaglutide was subjected to the stress conditions like acidic, basic, oxidative, photolytic and thermal conditions. In acidic, alkaline and peroxide stress conditions. In acidic, alkaline, and oxidative conditions degradant peaks were observed but there is no interference with semaglutide peak in all accelerated conditions, hence proving the stability indicating and specific nature of method. Assay result was found to be 99.99% w/w, thus it is proving that the method can also be applied for the estimation of semaglutide in pharmaceutical drug products.

4. LC-MS/MS method:

The objective is for the development of a specific and simple method to trace semaglutide presence in active pharmaceutical ingredients and pharmaceutical dosages.

This method was developed by Tae suk lee, Eun ji park, Minkyu chol, Hyun seok on, Yejin An, Taehyung kim, Tae soyoung shin and developed in the year 28 march 2023.

This study aimed to develop a novel liquid chromatograph tandem mass spectroscopy [LC-MS/MS] analysis of semaglutide in both plasma and brain to characterize the pharmacokinetics and brain distribution in rats. Semaglutide was extracted by simple protein precipitation with methanol from plasma and by solid phase extraction from brain tissue. Liraglutide was used as an internal standard. Gradient elution profiles with mobile phase comprising 0.1% formic acid in water and acetonitrile were used for chromatographic separation, The lower limit of qualification [LLOQ] of the LC-MS/MS assay was 0.5mg/ml for both eat plasma and brain. Intra & inter day accuracy ranged 89.20 – 109.50% in the plasma and 92.00 – 105.00% in the brain. Precision was within 8.92% in the plasma &7.44% in the brain.

Sprangue – Dawley rats were given semaglutide by intravenous [IV 0.02 MG/KG] & subcutaneous [sc 0.1&0.2mg/kg] injection. Plasma concentration of semaglutide showed a muti exponential decline with an average half – life  of 7.22-9.26 hours in rats. The subcutaneous bioavailability of semaglutide was 76.65 – 82.85%. The brain tissue of plasma partition co-efficient (Kp) value of semaglutide was estimated as <0.01. Among the different regions of the brain semaglutide concentrations were significantly higher in the hypothalamus. The analytical method and pharmacokinetic information may be helpful towards a better understanding of the effect of semaglutide in the brain and further development of GLP – 1 analogues for various brain disease.

5. LC-HRMS METHOD:

This method was developed by N. Arabouche, A. Blanchot, J.S.Raul, P. Kintz in the year 15 june 2024.

In this study the author have developed and validated a method for the identification and quantification of semaglutide in the whole blood using a LC-HRMS.

After the addition of the internal standard (bovine insulin), the blood was subjected to precipitation using a mix oof acetonitrile/methanol (70:30 , v:v). The validation procedure demonstrated an acceptable linearity between 28500µg/ml LOD and LOQ were 1and 2µg/ml respectively .Intra and inter day precision were below 20% at three concentrations. The method was successfully applied to blood samples of 3 diabetic patients under treatment of semaglutide. The samples tested positive with concentrations ranging from 31 to 70µg/ml. Which fall with in in the limits of therapeutic blood concentrations described in the literature.

6. UPLC-HR-SIM Method :

The method was developed by xinyi jiang,  jong zi, meng yu, yiran zhao,xiangyi wang, yanhe zhou, xinmiao Guo, jiuming he , jianpeng huang  in the year 2025.

To develop a highly sensitive and interference resistant ultra performance liquid chromatography (UPLC) coupled with high resolution selected ion monitoring (HR-SIM) mass spectrometry method for the qualification of semaglutide in biological samples and to apply it for pharmacokinetic analysis.

METHOD: The UPLC-HR-SLM method was utilized to quantify semaglutide in beagle plasma requiring minimal  sample pretreatment . The method demonstrated a lower limit of quantification (LLOQ) of 5.0µg/ml for semaglutide in beagle plasma .

RESULTS: Following intravenous (IV) administration at a dose of 0.030mg/kg, the plasma concentration of semaglutide in beagles exhibited a multi exponential decay pattern with an average elimination half-life (t1/2) of 44.90 ± 11.45 h.