Table 3.
Liquid chromatography-based techniques for quantitative determination of incretins in biological matrices and API formulation matrices regarding TDM programs, preclinical assessment, pharmacokinetic studies, entrapment efficiency and quality control protocols.
| Incretin | Matrix | Sample volume (μL) | Technique | Detection | LOD (pmol/L) | Column | Mobile phase |
Flow (μL/min) | Ref. | |||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Organic phase | Aqueous phase | Elution | Composition | |||||||||
| DPP-IV antagonists | Krill | 100 | HPLC | QTOF-MS | – | C18 | MeOH | Water | Isocratic | – | 10.00 | [43] |
| DPP-IV antagonists | Hydrolysate | – | HPLC | MS/MS | – | C18 | ACN: TFA (B) | TFA (A) | Gradient | 0–60.00 min: 0–45% B | 200.00 | [58] |
| DPP-IV antagonists | BMPH | – | HPLC | UV | – | C18 | ACN (B) | 0.01% TFA (A) | Gradient | 0–54.00 min: 0–60% B | 1500.00 | [59] |
| Exenatide | Hydrogel | 500 | HPLC | – | – | C4 | 85 mmol PBS: ACN (61: 39, v/v) (B) | 85 mmol PBS: ACN (95: 5, v/v) (A) | Gradient | 0–6.00 min: 65%–72% B 6.00–33.00 min: 72% B | 1000.00 | [52,60] |
| Exenatide | Microspheres | 100 | HPLC | MS/MS | – | C18 | ACN: 0.01% TFA (B) | 0.01% TFA (A) | Gradient | 0–33.00 min: 30%–44% B | 1000.00 | [61] |
| Exenatide | Microspheres | – | HPLC | UV | – | C18 | 0.1% TFA in ACN | 0.1% TFA | Isocratic | – | – | [49] |
| Exenatide | Microspheres | – | HPLC | UV | – | Protein specific | 0.1% TFA in ACN | 0.1% TFA in 2% sodium sulfate | Isocratic | – | 800.00 | [48] |
| Exenatide | Microspheres | – | HPLC | UV | – | C18 | ACN (B) | Water with 0.1% TFA (A) | Gradient | 0–16.00 min: 20%–60% B | 1000.00 | [46,50] |
| Exenatide | Microspheres | – | HPLC | UV | – | C18 | 80% ACN with TFA (B) | 0.1% TFA (A) | Gradient | 0–10.00 min: 20%–80% B 10.00–15.00 min: 80% B | 1000.00 | [62] |
| Exenatide | Microspheres | – | HPLC | UV | – | C18 | ACN: 0.01% TFA (B) | 0.01% TFA (A) | Gradient | 0–20.00 min: 30%–44% B | 1000.00 | [63] |
| Exenatide | Microspheres | – | HPLC | UV | – | C18 | ACN (A) | 0.05 M KH2PO4 (B) | Gradient | 0–20.00 min: 27%–43% A 20.00–20.10 min: 43%-27% B 20.10–28.00 min: 27% B | 1000.00 | [64] |
| Exenatide | Nanoparticles | 2000 | HPLC | UV | – | C18 | 80% ACN with 0.1% TFA (B) | Water with 0.1% TFA (A) | Gradient | 0–10.00 min: 20%–80% B 10.00–15.00 min: 80% B | 1000.00 | [65] |
| Exenatide | Nanoparticles | 10000 | HPLC | UV | – | C18 | 0.2% H3PO4: ACN | 0.2% H3PO4 | – | – | 500.00 | [29] |
| Exenatide | Nanoparticles | 200 | HPLC | UV | 477.71 | C18 | 0.1% TFA in ACN (B) | 0.1% TFA (A) | Gradient | 0–20.00 min: 42%–74% B | 500.00 | [27] |
| Exenatide | Porcine skin | – | HPLC | UV | 126593.47 | C4 | 0.1 M KH2PO4 in MeOH: 0.2 M ClNaO₄*H₂O in MeOH | Water | Isocratic | – | – | [16] |
| Exenatide | Solution | – | UPLC | QTOF-MS | – | C18 | 0.1% FA in ACN (B) | 0.1% FA (A) | Gradient | 0–2.00 min: 5% B 2.00–40.00 min: 5%–60% B 40.00–42.00 min: 60%–95% B 42.00–45.00 min: 95% B 45.00–50.00 min: 5% B |
200.00 | [66] |
| Exenatide | Solution | – | HPLC | MS | – | C18 | ACN: 0.01% TFA (B) | 0.01% TFA (A) | Gradient | 0–30.00 min: 30%–44% B | 1000.00 | [67] |
| Exenatide and glucagon | Human plasma | 1000 | LC | HRMS | – | C18 | ACN in MeOH (B) | 0.1% FA (A) | Gradient | 0–3.50 min: 15%–55% B 3.50–4.25 min: 55% B 4.25–5.50 min: 15% B | 500.00 | [68] |
| Exendin-4 | Eluate | – | UPLC | MS | – | C18 | 5% water: 0.1% FA: 2 mM NH4HCO2 in ACN (B) | 5% ACN: 0.1% FA: 2 mM NH4HCO2 (A) | Gradient | 0–5.00 min: 0–40% B 5.00–10.00 min: 40% B | 350.00 | [69] |
| Exendin-4 | Hydrolysate | – | HPLC | MS | – | C18 | ACN: 0.1% TFA (B) | – | Gradient | 0–35.00 min: 0–85% B | 1000.00 | [70] |
| Exendin-4 | Monkey plasma | 150 | UHPLC | MS/MS | 2388.56 | C18 | ACN (B) | 0.1% FA (A) | Gradient | 0–2.50 min: 25%–60% B 2.50–2.60 min: 60% B 2.60 min: 25% B | 1000.00 | [71] |
| Exendin-4 | Solution | – | HPLC | UV | – | C5 | ACN: water (70: 30, v/v) with 10 mM TFA (B) | 10 mM TFA A | Gradient | 0–2.00 min: 35% B 2.00–9.00 min: 35%–100% B | 2000.00 | [72] |
| Exendin-4 | Solution | – | HPLC | UV | – | C18 | ACN | 0.1% TFA | Isocratic | – | 500.00 | [73] |
| Exendin-4 analog | Rat retina | 10 | HPLC | FL | – | C18 | MeOH (B) | 0.1 M KH2PO4: 35% MeOH: 2% THF (A) | Gradient | 0–12.00 min: 0–40% B 12.00–17.00 min: 40% B 17.00–22.00 min: 0% B | 1000.00 | [74] |
| Exendin | Human plasma | 100 | UHPLC | MS/MS | 385.78 | C18 | 0.1% FA in ACN (B) | 0.1% FA (A) | Gradient | 0–12.00 min: 20%–65% B | 0.45 | [36] |
| 12.00–15.00 min: 85% B | 0.70 | |||||||||||
| 15.00–21.00 min: 20% B | 0.45 | |||||||||||
| GIP (1‐42) GIP (3‐42) |
Human plasma | 1000 | IM-LC | MS | 5.54 5.24 |
C18 | MeOH: water (90: 10, v/v) with 0.1% FA (A) | MeOH: water (10: 90, v/v) with 0.1% FA (B) | Gradient | 0–10.00 min: 100% B | 40.00 | [75] |
| 10.00–15.00 min: 100%-30% B | 40.00 | |||||||||||
| GLP-1 (7‐36) GLP-1 (9‐36) |
5.84 | 15.00–30.00 min: 100% A | 20.00 | |||||||||
| 30.00–35.00 min: 100% B | 40.00 | |||||||||||
| GLP-1 | Solution | 10 | HPLC | UV | – | C18 | ACN: 0.1% TFA (B) | Water: ACN: 0.1% TFA (A) | Gradient | 0–60.00 min: 40%–100% B | 500.00 | [76] |
| GLP-1 | GLP-1/HSA | – | HPLC | UV | – | Protein specific | 0.15 M sodium chloride | 0.02 M sodium phosphate | – | – | 800.00 | [53] |
| GLP-1 analogs | Mice plasma | 100 | LC | MS/MS | 10.00 | C18 | ACN with 0.1% FA (B) | Water: ACN (90: 10, v/v) (A) |
Gradient | 0–0.20 min: 5%–20% B 0.20–1.70 min: 20%–45% B 1.70–1.80 min: 40%–100% B 1.80–2.40 min: 100% B 2.40–3.00 min: 5% B |
500.00 | [9] |
| GLP-1 analogs | Mice serum | – | 2DLC | MS | – | C4 | ACN with 0.2% FA, 0.05% TFA and 10% 2-propanol (B) | Water with 0.2% FA, 0.05% TFA and 10% 2-propanol (A) | Gradient | 0–39.00 min: 20% B 39.00–79.00 min: 20%–90% B |
50.00 | [35] |
| Glucagon | Human plasma | 400 | UHPLC | MS/MS (SRM) |
7.18 | C18 | 0.2% FA in ACN (A) | 0.2% FA (B) | Gradient | 0–2.00 min: 22%–32% A 2.00–3.00min: 95% A 3.00–7.00 min: 22% A |
800.00 | [77] |
| Glucagon | Human plasma | 500 | IA-LC | MS/MS | 0.78 | C18 | 0.1% FA in ACN (B) | 0.1% FA (A) | Gradient | 0–2.00 min: 2% B | 15.00 | [37] |
| GLP-1 | 0.79 0.84 |
2.00–6.00 min: 20%–50% B 6.00–9.00 min: 50%–95% B 9.0–011.00 min: 95% B 11.00–13.50 min: 2% B |
4.00 | |||||||||
| Insulin | Human plasma | 500 | HPLC | MS/MS | 16.49 | C4 | 0.1% FA in ACN (B) | 0.1% FA (A) | Gradient | 0–2.50 min: 15%–40% B | 200.00 | [26] |
| – | 500.00 | |||||||||||
| Insulin Lixisenatide Exenatide Liraglutide Semaglutide |
Rat, minipig and human subcutaneous tissue | – | LC | HRMS (DDM) |
– | C18 | 0.1% FA in ACN (B) | 0.1% FA (A) | Gradient | 0–0.50 min: 0.5% B 0.50–5.00 min: 80% B 5.00–6.00 min: 80% B 6.00–6.10 min: 0.5%B 6.10–7.00 min: 0.5% B |
400.00 | [24] |
| Human plasma | 50 | – | ||||||||||
ACN, acetonitrile. BMPH, barbel muscle protein hydrolysate. ClNaO4*H2O, sodium perchlorate monohydrate. FA, formic acid. GIP, glucose-dependent insulinotropic peptide. GLP-1/HAS, glucagon-like peptide 1/human serum albumin fusion protein. HRMS, high resolution mass spectrometry. HRMS (DDM), high-resolution mass spectrometry data-dependent monitoring. H3PO4, phosphoric acid. IA-LC, immunoaffinity liquid chromatography. IM-LC, immunoprecipitation liquid chromatography. KH2PO4, monopotassium phosphate. LOD, limit of detection. MeOH, methanol. MS/MS, tandem mass spectrometry. MS/MS (SRM), tandem mass spectrometry selected reaction monitoring. NH4HCO2, ammonium formate. PBS, phosphate buffer solution. PDA, photodiode array. QTOF-MS, quadrupole time of flight mass spectrometry. TFA. trifluoracetic acid. THF, tetrahydrofuran. UPLC, ultra-performance liquid chromatography. 2DLC, 2-dimensional liquid chromatography.