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. 2019 Feb 26;9(3):143–155. doi: 10.1016/j.jpha.2019.02.001

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.