Insulin

Molecular formula: C 258 H 383 N 65 O 77 S 6 (human)

Molecular weight: 5807.6 (human) CAS Registry No.: 9004-10-8 (injection), 8049-62-5 (zinc suspension), 11061-68-0 (human), 12584-58-6 (pig), 11070-73-8 (cow), 9004-14-2 (neutral insulin), 8049-62-5 (isophane insulin), 9004-17-5 (protamine zinc suspension)

SAMPLE Matrix: blood, tissue Sample preparation: Pancreas. 1 g Tissue + 25 mL 6% trichloroacetic acid, homogenize, centrifuge, wash with 1 mL 5% trichloroacetic acid, extract precipitate twice at 37 by shaking for 2 h with 4 mL acid ethanol (A), adjust pH to 8.5-9 with concentrated ammonium hydroxide, inject 200 |xL aliquot. (Acid ethanol (A) was 15 mL EtOH + 5 mL water + 3 mL concentrated HCl.) Plasma. 1 mL Plasma + 2 mL water + 7.5 mL cold acid ethanol (B), stand at 4 for 12 h, centrifuge at 2800 rpm at 4 for 20 min, adjust pH of supernatant to 8.3 with concentrated ammonium hydroxide, keep at 4 for 15 min, centrifuge at 2800 rpm at 4 for 20 min, adjust pH of supernatant to 5.3 with 4 M HCl, for each 1 mL add 25 |xL 2 M ammonium acetate, readjust pH to 5.3, to each 10 mL slowly add 15 mL cold EtOH and 25 mL diethyl ether, keep at 4 for 12 h, centrifuge at 2800 rpm at 4 for 30 min. Remove precipitate and dry it under nitrogen gas. Dissolve in 100 mM pH 3.10 NaH2PO4, inject aliquot. (Acid ethanol (B) was 375 mL 95% EtOH and 7.5 mL concentrated HCl.) HPLCVARIABLES Column: 250 X 4.5 Spherisorb S5 ODS2 Mobile phase: Gradient. A was MeCN. B was 100 mM pH 3.10 NaH2PO4. A:B from 0:100 to 28:72 over 14 min, to 28.8:71.2 over 8 min, to 29.2:71.8 over 10 min, to 39.2:61.8 over 5 min, to 60:40 over 4 min. Flow rate: 1 Injection volume: 200 Detector: UV 220 CHROMATOGRAM Retention time: 33 (human), 35 (porcine) KEYWORDS pancreas; serum; human; pig REFERENCE
Knip, M. Analysis of pancreatic peptide hormones by reversed-phase high-performance liquid chromatography. Horm.Metab.Res., 1984, 16, 487-491

SAMPLE Matrix: bulk Sample preparation: Dissolve in mobile phase at 100-200 (xg/mL, inject 100 |xL aliquot. HPLCVARIABLES Guard column: 20 X 4.6 3 jxm Supelcosil LC-18DB ODS Column: 150 X 4.6 3 fim Supelcosil LC-18DB ODS Mobile phase: Gradient. A was 500 mM sodium sulfate + 300 mM NaH2PO4 adjusted to pH 2.5 with perchloric acid. B was water. C was MeCN:water 60:40. A:B:C at 20:30: 50 for 3 min then to 20:22.5:57.5 over 27 min. Column temperature: 45

Flow rate: 1 Injection volume: 100 Detector: UV 210

CHROMATOGRAM Retention time: 18 (bovine), 19.5 (bovine MDA), 21 (human), 22 (porcine), 23 (human MDA), 24 (porcine MDA) KEYWORDS cow; pig; human REFERENCE
Janssen, RS.L.; van Nispen, J.W.; van Zeeland, M.J.M.; Melgers, P.A.T.A. Complementary information from isotachophoresis and high-performance liquid chromatography in peptide analysis. J.Chromatogr., 1989, 470, 171-183

SAMPLE Matrix: formulations Sample preparation: Dissolve 10 mg microspheres in 2 mL MeCN, centrifuge at 3000 rpm for 5 min, discard MeCN, repeat process three times, dissolve pellet in 5 mL pH 7.4 Tris buffer containing 0.1% trifluoroacetic acid, inject a 20 |xL aliquot. HPLCVARIABLES Column: 150 X 6 Asahipak ODP-50 6D (Asahi Chemical) Mobile phase: MeCN: buffer 28:72 (Buffer was 0.3% ethanolamine adjusted to pH 2.0 with phosphoric acid.) Column temperature: 40 Flow rate: 2 Injection volume: 20 Detector: UV 220 CHROMATOGRAM Retention time: 7 OTHER SUBSTANCES Simultaneous: degradation products KEYWORDS microspheres; cow REFERENCE
Uchida, T.; Yagi, A.; Oda, Y.; Nakada, Y; Goto, S. Instability of bovine insulin in poly(lactide-co-glycolide) (PLGA). Chem.Pharm.BulL, 1996, 44, 235-236

SAMPLE Matrix: formulations Sample preparation: 100 |xL Injection + 50 |xL 50 |xg/mL benzoic acid in water + 250 |xL water, vortex briefly, inject a 50 |xL aliquot. HPLCVARIABLES Column: 250 X 4.6 5 |xm 300 A silica for proteins and peptides (Vydac) Mobile phase: MeCN: 50 mM pH 2.4 KH2PO4 25:75

Flow rate: 1 Injection volume: 50 Detector: UV 230

CHROMATOGRAM Retention time: 11.16 Internal standard: benzoic acid OTHER SUBSTANCES Simultaneous: degradation products KEYWORDS injections; stability-indicating REFERENCE
Hoyer, G.L.; Nolan, RE., Jr.; LeDoux, J.H.; Moore, L.A. Selective stability-indicating high-performance liquid chromatographic assay for recombinant human regular insulin. J.Chromatogr.A, 1995, 699, 383-388

SAMPLE Matrix: formulations Sample preparation: Prepare a 150 |xg/mL solution in 10 mM HCl, inject a 20 jxL aliquot. HPLC VARIABLES Column: 250 X 4.6 5 |xm Ultremex octadecylsilane Mobile phase: MeCN: buffer 26:74 (Buffer was 200 mM sodium sulfate adjusted to pH 2.3.) Column temperature: 40 Flow rate: 0.9 Injection volume: 20 Detector: UV 214 CHROMATOGRAM Retention time: 17.5 KEYWORDS injections REFERENCE
Lookabaugh, M.; Biswas, M.; Krull, LS. Quantitation of insulin injection by high-performance liquid chromatography and high-performance capillary electrophoresis. J.Chromatogr., 1991, 549, 357-366

SAMPLE Matrix: formulations HPLCVARIABLES Column: 50 X 4.6 C4 wide pore (300 A) (Supelco) Mobile phase: Gradient. A was MeOH: isopropanol: water 4:1:95 containing 2.8 g/L NaCl. B was MeOH:isopropanol:water 60:10:30 containing 4.2 g/L NaCl. A:B from 45:55 to 30:70 over 5 min. Flow rate: 1 Injection volume: 20 Detector: E, Bioanalytical systems Model LC-4B, dual glassy-carbon working electrode used in parallel mode, +0.65 V and +0.80 V (monitored), stainless steel auxiliary electrode, Ag/AgCl reference electrode following post-column reaction. The column effluent flowed at 0-5 through a 2 mL knitted coil of 0.5 mm i.d. PTFE tubing irradiated with a low pressure mercury lamp (Photronix Model 816) to the detector. CHROMATOGRAM Retention time: 8

OTHER SUBSTANCES

Also analyzed: b-lactoglobulin A, lysozyme, phenylalanine, ribonuclease A, tryptophan, tyramine

KEYWORDS

post-column reaction
REFERENCE
Dou, L.; Krull, LS. Determination of aromatic and sulfur-containing amino acids, peptides, and proteins using high-performance liquid chromatography with photolytic electrochemical detection. Anal.Chem., 1990, 62, 2599-2606

SAMPLE Matrix: solutions HPLCVARIABLES

Column: 150 X 4.6 Synchropak C4 Mobile phase: Gradient. A was 0.05% trifluoroacetic acid in water. B was 0.05% trifluoroacetic acid in MeCN. A:B from 74:26 to 38:62 over 15 min. Flow rate: 1.5 Detector: UV 220
CHROMATOGRAM Retention time: 4.7 REFERENCE
Ho, H.-O.; Hsiao, C-C; Sheu, M.-T. Preparation of microemulsions using polyglycerol fatty acid esters as surfactant for the delivery of protein drugs. J.Pharm.Sci., 1996, 85, 138-143

SAMPLE Matrix: solutions HPLCVARIABLES

Column: 150 X 4.6 5 pirn Zorbax 300 A SB-C3 Mobile phase: Gradient. A was MeCN: water: trifluoroacetic acid 5:95:0.1. B was MeCN: water:trifluoroacetic acid 5:95:0.085. A:B from 85:15 to 47:53 over 20 min. Column temperature: 35 Flow rate: 1 Injection volume: 10 Detector: UV 215
CHROMATOGRAM Retention time: 10 OTHER SUBSTANCES

Simultaneous: angiotensin II, carbonic anhydrase, cytochrome C, leucine enkephalin, lysozyme, myoglobin, RNAase
REFERENCE
Ricker, R.D.; Sandoval, L.A.; Permar, B.J.; Boyes, B.E. Improved reversed-phase high performance liquid chromatography columns for biopharmaceutical analysis. J.Pharm.Biomed.AnaL, 1996, 14, 93-105

SAMPLE Matrix: solutions

HPLCVARIABLES Column: 150 X 4.6 Develosil ODS-HG-5 (Nomura Chemical) Mobile phase: MeCN: 100 mM pH 9.0 phosphate buffer 26:74 Column temperature: 40 Flow rate: 0.8 Detector: UV 214 CHROMATOGRAM Retention time: 18 OTHER SUBSTANCES Simultaneous: degradation products REFERENCE
Yomota, C; Yoshii, Y.; Takahata, T.; Okada, S. Separation of B-3 monodesamidoinsulin from human insulin by high-performance liquid chromatography under alkaline conditions. J.Chromatogr.A, 1996, 721, 89-96

SAMPLE Matrix: solutions HPLCVARIABLES Column: 250 X 4.6 Protein & Peptide C18 (Vydac) Mobile phase: MeCN:buffer 26:74 (Buffer was 28.4 g sodium sulfate and 2.7 mL phosphoric acid in 1 L water, pH adjusted to 2.3 with ethanolamine (if necessary).) Column temperature: 40 Flow rate: 0.8 Detector: UV 214 CHROMATOGRAM Retention time: 15 OTHER SUBSTANCES Simultaneous: degradation products REFERENCE
Yomota, C; Yoshii, Y; Takahata, T.; Okada, S. Separation of B-3 monodesamidoinsulin from human insulin by high-performance liquid chromatography under alkaline conditions. J.Chromatogr.A, 1996, 721, 89-96

SAMPLE Matrix: solutions HPLCVARIABLES Column: 150 X 4 Armsorb-Si-300 p (DM) (Armchrom, Yerevan, Armenia) Mobile phase: Gradient. A was MeCN :1 M ammonium acetate 10:90. B was MeCN :1 M ammonium acetate 50:50. A:B from 76:24 to 66:34 over 40 min. Flow rate: 0.8 Detector: UV 200 CHROMATOGRAM Retention time: 26 OTHER SUBSTANCES Simultaneous: proinsulin

KEYWORDS

recombinant; comparison with capillary electrophoresis

REFERENCE
Klyushnichenko, V.E.; Koulich, D.M.; Yakimov, S.A.; Maltsev, K.V.; Grishina, G.A.; Nazimov, LV.; WuIfson, A.N. Recombinant human insulin. III. High-performance liquid chromatography and high-performance capillary electrophoresis control in the analysis of step-by-step production of recombinant human insulin. J.Chromatogr.A, 1994, 661, 83-92

SAMPLE

Matrix: solutions
HPLCVARIABLES

Column: 600 X 7.5 TSK G 2000 SW (TOSOH) Mobile phase: MeCN: buffer 5:95 (Buffer was 100 mM pH 7.0 phosphate buffer containing 200 mM sodium sulfate.) Flow rate: 1 Detector: UV 200
CHROMATOGRAM

Retention time: 19
OTHER SUBSTANCES

Simultaneous: proinsulin
KEYWORDS

recombinant; comparison with capillary electrophoresis; SEC

REFERENCE
Klyushnichenko, V.E.; Koulich, D.M.; Yakimov, S.A.; Maltsev, K.V.; Grishina, GA.; Nazimov, I.V.; WuIfson, A.N. Recombinant human insulin. III. High-performance liquid chromatography and high-performance capillary electrophoresis control in the analysis of step-by-step production of recombinant human insulin. J.Chromatogr.A, 1994, 661, 83-92

SAMPLE

Matrix: solutions Sample preparation: Prepare a 1 mg/mL solution in MeCN.water 20:80, inject a 10 J U L L aliquot.
HPLCVARIABLES

Column: Nucleosil 100-5 C18 RP Mobile phase: Gradient. MeCN: buffer from 20:80:40:60 over 20 min. (Buffer was 83 mM phosphoric acid adjusted to pH 2.25 with triethylamine.) Injection volume: 10 Detector: UV 215
CHROMATOGRAM

Retention time: 15
REFERENCE
Lenz, V.J.; Gattner, H.-G.; Leithauser, M.; Brandenburg, D.; Wollmer, A.; Hocker, H. Proteolyses of a fluorogenic insulin derivative and native insulin in reversed micelles monitored by fluorescence emission, reversed-phase high-performance liquid chromatography, and capillary zone electrophoresis. Anal.Biochem., 1994, 221, 85-93

SAMPLE

Matrix: solutions
HPLCVARIABLES

Column: 250 X 4.5 5 ixm Kromasil C8 (Eka-Nobel) Mobile phase: Gradient. A was MeCN: water 10:90 containing 0.1% trifluoroacetic acid. B was MeCN:water 90:10 containing 0.1% trifluoroacetic acid. A:B from 0:100 to 75:25 over 8 min, to 25:75 over 12 min. Flow rate: 2 Detector: UV 254
CHROMATOGRAM

Retention time: 10.7

OTHER SUBSTANCES

Simultaneous: angiotensin I, angiotensin II, bradykinin, leucin enkephalin, lysozyme, melittin, methionine enkephalin, oxytocin
REFERENCE
Bodman Product Guide, Bodman, Aston PA, 1992, p. 104

SAMPLE

Matrix: solutions
HPLCVARIABLES

Column: 150 X 4.1 10 \xm PRP-3 (Hamilton) Mobile phase: Gradient. A was 0.1% trifluoroacetic acid in 50 mM NaOH. B was 0.1% trifluoroacetic acid in MeCN. A:B from 100:0 to 40:60 over 30 min. Flow rate: 2 Detector: UV 220
CHROMATOGRAM

Retention time: 14
OTHER SUBSTANCES

Simultaneous: cytochrome C, lysozyme, myoglobin, ribonuclease A, trypsin

REFERENCE
Rainin Catalog 1991-2, Rainin Instrument Co., Woburn MA, 1991, p. 3.33

SAMPLE

Matrix: solutions
HPLCVARIABLES

Column: 250 X 4.6 PLRP-S IOOOA (Polymer Labs) Mobile phase: Gradient. A was 0.1% trifluoroacetic acid in water. B was 0.1% trifluoroacetic acid in 95% MeCN. A:B from 80:20 to 40:60 over 22 min. Flow rate: 1.5 Detector: UV 220
CHROMATOGRAM

Retention time: 5

OTHER SUBSTANCES Simultaneous: bovine serum albumin, cytochrome C, lysozyme, myoglobin, ovalbumin, ribonuclease REFERENCE Rainin Catalog 1991-2, Rainin Instrument Co., Woburn MA, 1991, p. 3.63 SAMPLE Matrix: solutions Sample preparation: Dissolve 70 mg trinitrobenzenesulfonic acid in 1 mL 100 mM pH 8.2 sodium bicarbonate and immediately add an aliquot to 50 volumes of 10 mg/mL insulin in 100 mM pH 8.2 sodium bicarbonate, let stand in the dark at room temperature for 2 h, add to a 150 X 60 column of Sephadex G25 made up in 100 mM pH 8.2 sodium bicarbonate, collect the major colored band and lyophilize it. Reconstitute, inject an aliquot. HPLCVARIABLES Column: ixBondapak C18 Mobile phase: Gradient. MeCN: 100 mM pH 3.6 sodium phosphate 25:75 for 10 min, to 45:55 over 1 h Flow rate: 1 Detector: UV 280 CHROMATOGRAM Retention time: 50 KEYWORDS derivatization REFERENCE Wallace, G.R.; McLeod, A.; Chain, B.M. Chromatographic analysis of the trinitrophenyl derivatives of insulin. J.Chromatogr., 1988, 427, 239-246

ANNOTATED BIBLIOGRAPHY Lakhiari, H.; Legendre, E.; Muller, D.; Jozefonvicz, J. High-performance affinity chromatography of insulin on coated silica grafted with sialic acid. J.Chromatogr.B, 1995, 664, 163173 Calvaruso, G.; Tesoriere, G.; Vento, R.; Giuliano, M.; Carabillo, M. High-performance liquid chromatographic method for the determination of insulin synthesis in biological systems. J.Chromatogr.B, 1994, 660, 259-264 [SEC; GPC; reverse phase] Dimov, N.; Simeonov, S. Experimental models for optimization of insulin separation on reversed phase columns. Biomed.Chromatogr., 1994, 8, 32-36 [gradient; cow; pig] Kliushnichenko, V.E.; Iakimov, S.A.; Arutiunian, A.M.; Ivanov, A.E.; Mal'tsev, K.V.; Vul'fson, A.N. [Genetic engineering of human insulin. IV. Development and optimization of an analysis system using reversed phase high pressure liquid chromatography]. Bioorg.Khim., 1994, 20, 1080-1088 Klyushnichenko, V.E.; Yakimov, S.A.; Arutyunyan, A.M.; Ivanov, A.E.; Maltsev, K.V.; Wulfson, A.N. Recombinant human insulin V. Optimization of the re versed-phase high-performance liquid chromatographic separation. J.Chromatogr.B, 1994, 662, 363-369 [gradient] Ohkubo, T. High performance liquid chromatographic analysis of polypeptide hormones in transplanted rat islets. Biomed.Chromatogr., 1994, 8, 301-305 Yamamoto, A.; Taniguchi, T.; Rikyuu, K.; Tsuji, T.; Fujita, T.; Murakami, M.; Muranishi, S. Effect of various protease inhibitors on the intestinal absorption and degradation of insulin in rats. Pharm.Res., 1994, 11, 1496-1500 [rat; gradient] Salem, LL; Bedmar, M.C.; Medina, M.M.; Cerezo, A. Insulin evaluation in Pharmaceuticals: Variables in RP-HPLC and method validation. J.Liq.Chromatogr., 1993, 16, 1183-1194 [formulations]

Cruz, N.; Lopez, M.; Estrada, G.; Alvarado, X.; de Anda, R.; Balbas, P.; Gosset, G.; Bolivar, F. Preparative isolation of recombinant human insulin-A chain by ion exchange chromatography. J.Liq.Chromatogr., 1992, 15, 2311-2324 Cruz, N.; Antonio, S.; de Anda, R.; Gosset, G.; Bolivar, F. Preparative isolation by high performance liquid chromatography of human insulin B chain produced in Escherichia coli. J.Liq.Chromatogr., 1990, 13, 1517-1528 Caprioli, R.M.; DaGue, B.; Fan, T.; Moore, W.T. Microbore HPLC/mass spectrometry for the analysis of peptide mixtures using a continuous flow interface. Biochem.Biophys.Res.Commun., 1987, 146, 291-299 [LC-MS; microbore; gradient; UV detection; cow; sheep; pig; horse] Schrader, E.; Pfeiffer, E.F. The influence of motion and temperature upon the aggregational behaviour of soluble insulin formulations investigated by high performance liquid chromatography. J.Liq.Chromatogr., 1985, 8, 1139-1157 [GPC; SEC; reverse-phase; pig; human] Schrader, E.; Pfeiffer, E.F. HPLC-gel nitration of insulin during short and longtime infusion by artificial delivery systems. J.Liq.Chromatogr., 1985, 8, 1121-1137 [GPC; SEC] Smith, H.W., Jr.; Atlins, L.M.; Binkley, D.A.; Richardson, W.G.; Miner, D.J. A universal HPLC determination of insulin potency. J.Liq.Chromatogr., 1985, 8, 419-439 [cow; pig; human] Ohta, M.; Tokunaga, H.; Kimura, T.; Satoh, H.; Kawamura, J. Analysis of insulins by high-performance liquid chromatography. III. Determination of insulin in various preparations. Chem.Pharm.Bull., 1984, 32, 4641-4649 Ohta, M.; Tokunaga, H.; Kimura, T.; Yamaha, T. [Analysis of insulin by high-performance liquid chromatography. IV. Stability of insulin in hydrochloric acid]. Yakugaku Zasshi, 1984, 104, 1309-1313 Ohta, M.; Tokunaga, H.; Kimura, T.; Satoh, H.; Kawamura, J. [Analysis of insulin in preparations by high performance liquid chromatography]. Yakugaku Zasshi, 1982, 102, 1092-1094 Pocker, Y; Biswas, S.B. A simple liquid chromatographic method for analysis of insulin and its derivatives. J.Liq.Chromatogr., 1982, 5, 1-14 [SEC; GPC]