Analytica Chimica Acta 553 (2005) 64–72

Application of electrospray ionization-mass spectrometry to screenextractants for determination of insulin in an emulsion

system by HPLC-UV

Yong Luo, Kaixun Huang, Huibi Xu∗

Department of Chemistry, Huazhong University of Science & Technology, 1037 Luoyu Road, Wuhan 430074, PR China

Received 2 June 2005; received in revised form 27 July 2005; accepted 4 August 2005Available online 5 October 2005

Abstract

Soybean lecithin and phenol intensely interfered in the determination of insulin with HPLC-UV in an emulsion system which was developedfor transmucosal delivery. Thus, in this study electrospray ionization mass spectrometry (ESI-MS) with ion trap detection was applied toscreen extractants to remove soybean lecithin and phenol in an insulin-loaded emulsion system (IES). The results showed thatn-pentanol was a

aneously, andcludingprecisionacy ando study thent

ryan

calwas

rob-alityriticalthod

ntod fortect-edy

liq--

suited extractant to meet the measurement requests because a large quantity of soybean lecithin and phenol were removed simultthere appeared no interferences brought byn-pentanol in insulin measurement. The analytical method was assessed by the criteria inlinearity (linearity range: 1.05–7.34 U/mL and correlation coefficient: 0.99936), accuracy (recoveries: 98.1–102.5%), within-day(R.S.D.: 0.5%,n = 6), LOD (0.01 U/mL), and LOQ (0.03 U/mL), and exhibited high sensitivity and specificity, and considerable accurprecision on the determination of insulin in the emulsion system. The application of this method had been successfully performed tstorage conditions of IES, and the results indicated that insulin in IES would be stable at 4◦C in light proof condition, which was significafor further pharmaceutical research.© 2005 Elsevier B.V. All rights reserved.

Keywords: ESI-MS; Screening extractants; Insulin; Emulsion system; HPLC

1. Introduction

Insulin is a protein hormone synthesized by pancreatic�-cells and used in controlling the blood-glucose level. It isstored in�-cells as a two-Zn2+-containing hexamer. Insulinmonomer contains 51 amino acid residues in two chains(chain A with 21 residues and chain B with 30 residues)linked together by two disufide bridges[1,2]. The sequenceof human insulin is shown inFig. 1.

Since its discovery in 1921, insulin has remained a majorclinic drug for treatment of diabetes mellitus (IDDM andNIDDM) [3]. However, insulin injection can bring so greatpain and much discomfort to the diabetics that non-injectabledelivery routes have attracted many investigations of pharma-

∗ Corresponding author. Tel.: +86 27 87543532; fax: +86 27 87543632.E-mail addresses: dennyluo@sohu.com (Y. Luo),

hbxu@mail.hust.edu.cn (H. Xu).

ceutical researchers[4–13]. We have studied a new delivemethod of insulin for nearly 10 years, and developedeffective insulin-loaded emulsion system (IES) for buctransmucosal delivery. The hypoglycaemic effect of IESsignificant and the bioavailability exceeded 20%[12]. To bea hypoglycaemic drug, some further pharmaceutical plems, such as the storage conditions, shelf life, and qustandards, had to be elucidated. In all these studies, the cpoint was to establish a rapid and accurate analytical mefor insulin identification and quantification.

A variety of analytical methods, roughly sorted iimmune and non-immune methods, had been applieinsulin detection. The former one had been used for deing insulin in vivo early from 1950s, which mainly includradioimmunoassay (RIA)[14–17], enzyme immunoassa(EIA) [18–20], and luminescent immunoassay (LIA)[21,22].The non-immune methods, especially high performanceuid chromatography (HPLC)[23–27] and capillary elec

0003-2670/$ – see front matter © 2005 Elsevier B.V. All rights reserved.doi:10.1016/j.aca.2005.08.010

Y. Luo et al. / Analytica Chimica Acta 553 (2005) 64–72 65

Fig. 1. The sequence of human insulin.

trophoresis (CE)[28–31], had been widely used for insulindetection in vivo and in vitro. As we known, non-immunemethod for insulin detection had insufficient sensitivity com-pared with immune method because of the interferencesfrom the sample matrix. However, HPLC could be a rapidand accuracy method, if proper pretreatment procedure wasapplied for reverse-phase HPLC, a typical analytical methodfor quantifying peptide and protein in liquid drug, owned thehigh resolution and easy automation to meet the sample testrequests.

Soybean lecithin, a very important pharmaceutical bioma-terial, commonly consisting of phosphotidyl choline (PC),phosphotidyl ethanolamine (PE), phosphotidyl inositol (PI),phosphotidyl serine (PS), sphingolipids, etc., has been widelyused in drug delivery system to act as absorption enhancerfor peptide and protein drug because of its high affinity withboth the loaded-drug molecule and biomembrane[32–34].But this high affinity of soybean lecithin leaded to decreaseof the separation efficiency of insulin from drug matrix andthe efficiency of C18 chromatographic column. Phenol wasusually used in pharmaceutical preparations as antimicrobial[35], and especially as stabilizer for insulin due to its abil-ity of forming a specific non-covalent complex with insulin[36–40]. But, phenol has considerable UV absorption, andit would possibly interfere in the UV absorption of peptideor protein. Therefore, an appropriate pretreatment method,w ithina pos-s

pa-bb per-i ucem find-i duref ulini ome-t dT h-n to bea ules,s ona ple-m tein,s fica-t d the

fundamental principle of chemistry, especially in solution byESI-MS[51–54]. In addition, the content of the analyte couldbe semi-quantitated by the absolute signal intensity while amass spectrograph with ion trap detection was applied as ascreening method[55].

In this paper, a suited extractant was selected by ESI-MS,and an analytical method for determination of insulin in IESby HPLC was set up. The application of the HPLC methodwas performed to study the storage conditions of IES.

2. Materials and methods

2.1. Materials

Recombinant human insulin (insulin, potency: 29.2 U/mg)was purchased from Sino-American Tonghua Antaike Bio-logics Engineering Co. Ltd. (Jilin, China), standard recombi-nant human insulin (standard insulin, potency: 26.2 U/mg)was obtained from National Institute for the Control ofPharmaceutical and Biological Products (Beijing, China).Redistilled phenol was purchased from Tianyuan ChemicalCompany (Wuhan, China), and soybean lecithin from Shang-hai Jinban Pharmaceutical Co. Ltd. (Shanghai, China). Otherchemicals were of HPLC or analytical grade, and used with-out further purification. Deionized water was purified in aM

2

s fol-l os-p /m)p d inp them , andt PBS( ar-i era g tog ina lin-le thes

2i

n l)w Eachw eanl ndE erep vide

hich could greatly reduce the content of soybean lecnd phenol but simultaneously remain insulin as more asible, should be performed before HPLC test.

Liquid–liquid extraction is easy to develop and cale of providing very clean sample extracts[41,42]. It haseen applied for emulsion system before analytical ex

ments[43–46]. The cleaner sample extracts could reduch burden on the chromatographic column. Hence,

ng a suitable extractant for IES would be a key proceor the establishment of the analytical method of insn IES. To obtain the suitable extractant, mass spectrry was applied as screening method[43]. Due to Fenn ananaka’s contribution to the novel “soft” ionization teciques in 2002, mass spectrometry has been developednew approach to study large, polar, non-volatile molecuch as insulin[47–50]. Moreover, by using a high-resolutinalyzer with broader mass-to-charge range, more comentary information is obtained to investigate intact pro

uch as confirmation of non-covalent interactions, identiion of the unknowns, research of molecular structure, an

illi-Q plus system from Millipore prior to use.

.2. Preparation of insulin-loaded emulsion system

The insulin-loaded emulsion system was prepared aowing steps: insulin powder was dissolved in pH 7.4 phhate buffer saline (PBS, 0.05 mol/L) containing 0.2% (mhenol as stock solution; soybean lecithin was dissolveropanediol as transmucosal enhancer of insulin, whileass ratio of lecithin and propanediol was set as 3:10

he resulting solution was added to a proper amount ofpH 7.4, 0.05 mol/L) under continuously stirring by a sheng machine for 30 min (10,000± 100 rpm). Then, a propmount of insulin stock solution was added while stirrinive a mixture with every milliliter containing 40 U of insulnd 0.015 g soybean lecithin. The formulation of the insu

oaded emulsion had been developed by Xu et al.[12]. Themulsion system without insulin (ES) was prepared iname way except that no insulin was loaded.

.3. Screening extractants for IES by electrosprayonization mass spectrometry (ESI-MS)

Eight alcohols (n-butanol,n-pentanol,n-hexanol,n-hepta-ol, n-octanol, n-nonanol, n-decanol, andn-hendecanoere selected to be the extractant candidates for IES.as mixed with ES by fully shaking to extract most soyb

ecithin from ES (excipient). The volume ratio of alcohol aS was kept at 2:1 (alcohol:ES). Then, the mixtures wlaced quiescently for 2.0 h, and the mixtures would di

66 Y. Luo et al. / Analytica Chimica Acta 553 (2005) 64–72

into alcohol phase and aqueous phase. The alcohol phase waspicked out and continuously pumped into the ESI source at aflow rate of 2.7�L/min using a Cole-Parmer 74900 syringepump (Cole-Parmer Instrument Company, Germany). Themass spectra were obtained by using a Bruker Esquire-LCion-trap mass spectrometer (Bruker Co., Germany) equippedwith a gas nebulizer probe capable of analyzing ions up tom/z 6000. The experiments were operated as follows: thenebulizer pressure was 11 psi, capillary voltage was typicallyat 7 kV, and heated capillary temperature was at 300◦C.Nitrogen was used as drying gas with a flow rate 4 L/min. Thescan ranges were fromm/z 50 to 2200 in positive-ion mode.

To show the extracted effect of insulin, insulin was dis-solved quantitatively in PBS (pH 7.4, 0.05 mol/L), and thenthe resulting insulin solution (40 U/mL) was mixed with theeight alcohols, respectively. The screening procedures fol-lowed the same methods as above (nebulizer pressure was18 psi and capillary voltage was typically at 9 kV).

The typical ESI-MS spectrum of soybean lecithin wasobtained by the following steps: quantitative soybean lecithinwas dissolved in chloroform (0.15%, m/m), and then theobtained solution was continuously pumped into the ESIsource under the same conditions of the ESI-MS. The typ-ical ESI-MS spectrum of insulin was obtained by pumpingthe standard insulin solution (40 U/mL, pH 2.0) into the ESIsource.

Dal-t liedB thep

2

withn ndn -t ouldd ase)Tm (pH7 tedst amem

2

atera olu-t tedt 5.24,6 .0).Tf toe urvew ns of

insulin. Peak area of insulin versus the corresponding con-centration was plotted as standard curve to show the linearityrange.

2.6. HPLC system and chromatographic conditions

The HPLC system was an Agilent 1100 series (AgilentTechnologies, USA), consisting of a G1311A quaternarypump, a G1379A degasser, a G1313A autosampler, a G1315Bdiode-array detector (DAD), and a G1316A column thermo-stat. Separation was performed at 30◦C on a 4.6 mm× 50 mmbase stable column packed with 5�m C18 reversed-phaseparticles. The column was obtained from Dalian Elite Analyt-ical Instrument Co. Ltd. (Dalian, China). Gradient elution wascarried out with 0.05 mol/L phosphate buffer saline (PBS, pH3.0, eluent A) and acetonitrile (eluent B). The gradient startedat 26% of B (v/v) and was increased linearly to reach 32% ofB in 6.0 min, then this volume ratio of A and B (A:B = 68:32,v/v) was maintained for 4.0 min, and the volume percent ofB was increased linearly to reach 38% from 32% in succes-sional 6.0 min. The post time was set at 6.0 min. The mobilephase was filtered through a 0.45�m pore size membranefilter before further use. The flow rate was set at 1.0 mL/min,the injection volume was 20�L, and detection wavelengthwas 214.4 nm. Data acquisition and analysis were performedby using online and offline of Aglilent ChemStation, respec-t

2

hro-m andE itht enceo

pro-d mesi t as7 h thev

per-c nt ofa lutedw inedi ),r

n-t and1

2s

ar-m espec-t

The signal intensity was obtained by using the Brukeronics Data Analysis 3.0 (Data Processor Software, Appiosystems). This software was also used to deal withrofile of the spectrum.

.4. Pretreatment of IES and ES before HPLC test

Based on the screening results, IES was mixed up-pentanol by fully shaking. The volume ratio of IES a-pentanol was kept at 1:2 (IES:n-pentanol). Then, the mixure was placed quiescently for 2.0 h, and the mixture wivide into two phases (alcohol phase and aqueous phhe aqueous phase was filtered through a 0.45�m pore sizeembrane filter, and the filtrate was diluted with PBS.4, 0.05 mol/L) by 10 times. At last the obtained diluolution was filled into the test vial, and placed at 4◦C forhe following test by HPLC. ES was pretreated with the sethod as above.

.5. The linearity range of insulin for HPLC test

Standard insulin was dissolved in the deionized wt pH 2.0 by adding hydrochloric acid to be a stock s

ion (36.68 U/mL), and then the stock solution was diluo a serial standard solutions (1.05, 2.10, 3.14, 4.19,.29, and 7.34 U/mL) by adding deionized water (pH 2he standard solutions of insulin were stored at 4◦C for the

ollowing use. Insulin biopotency unit (U/mL) was usedxpress insulin concentration in this paper. Standard cas prepared by using the above serial standard solutio

.

ively (for LC 3D Rev.A.09.01, 1206).

.7. Validation of analytical method

The specificity of the assay was evaluated by the catographic spectra of standard insulin solution, IES,S. The retention time of insulin in IES was compared w

hat of standard insulin solution. ES was tested for the absf insulin peak compared with the other two.

The method precision was evaluated by within-day reucibility assay. The pretreated IES was injected six ti

n 1 day, and the interval of every two injections was se5 min. The precision of the method was expressed witalues of R.S.D. of replicate measurements.

Accuracy of the assay methods was defined as theentage of recovery by the assay of the known amounalyte added in the sample. The prepared IES (diith PBS by 10 times) as samples precisely conta

nsulin of 3.2 (80%), 4.0 (100%), and 4.8 U/mL (120%espectively.

Determination of the limit of detection (LOD) and quaitation (LOQ) were performed while the S/N was at 3:10:1, respectively.

.8. The application of the analytical method for thetorage conditions of IES

To study the storage conditions of IES for further phaceutical researches, the prepared IES was stored, r

ively, at 4◦C for 0, 3, 6, 9, and 12 months; at 25◦C for

Y. Luo et al. / Analytica Chimica Acta 553 (2005) 64–72 67

0, 5, 10, 15, 20, 25, and 30 days; placed under intense light(2500± 500 lx) for 0, 1, 3, 5, 7, and 10 days[56]. These sam-ples were tested by HPLC-UV. The quantification of insulinin IES was performed with external standard method.

3. Results and discussion

3.1. The interferences caused by soybean lecithin andphenol in HPLC determination of insulin

IES was a complex emulsion system containing soybeanlecithin as absorption enhancer and phenol as antimicrobial[12], both of which could intensely interfere in the determi-nation of insulin by HPLC. Soybean lecithin, an amphipilicmolecule, could form vesicles of different sizes in IES, andwas likely to attach insulin to the formed vesicles[32–34].Because of the existence of soybean lecithin in IES, the sep-aration efficiency of insulin from drug matrix, and efficiencyof C18 chromatographic column would decrease, with thepressure of the chromatographic column going up very high.Phenol could possibly interfere in the determination of insulinby HPLC because of its considerable UV absorption. To showthe interferences caused by phenol under the optimized chro-matographic conditions, standard solutions of phenol (0.2%,m ings pec-

tively, to obtain the typical chromatographic spectra of insulinand phenol (Fig. 2). The main peaks of insulin and phe-nol did not overlap, and could be easily distinguished, butthe phenol peak exhibited a long tail which covered therange of insulin peak. The tailed peak of phenol had con-siderable area (1187.6,Fig. 2B) compared with insulin peak(3073.3,Fig. 2A), which made the area of insulin peak inFig. 2C (3728.1) bigger than that inFig. 2A (3073.3), result-ing in errors in insulin quantification intensively. The tailedpeak of insulin inFig. 2A was the degradation product ofinsulin (desamido B3 insulin) which had similar biopotencyas insulin, and no interference was found on insulin quan-tification[57]. Hence, soybean lecithin and phenol should beremoved from the system as much as possible before HPLCtest.

3.2. Screening extractants for HPLC test by ESI-MS

Soybean lecithin was dissolved in chloroform (0.15%,m/m), and then the resulting solution and insulin standardsolution (4 U/mL, pH 2.0) were continuously pumped intothe ESI source to obtain the typical ESI-MS spectra of soy-bean lecithin and insulin (Fig. 3). There were four main grouppeaks of soybean lecithin inFig. 3A, which were set as thestandard of screening soybean lecithin by ESI-MS. Insulin, am olu-t d in

Fs

/m) and insulin (4 U/mL), and a mixed solution containame quantity of insulin and phenol were injected, res

ig. 2. The chromatographic spectra of insulin and phenol. (A) Typical specolution containing insulin and phenol.

acro biomolecule, could easily carry protons in acid sion during electrospray ionization course, which resulte

trum of Rh-insulin, (B) typical spectrum of phenol, and (C) the spectrum ofthe

68 Y. Luo et al. / Analytica Chimica Acta 553 (2005) 64–72

Fig. 3. ESI-MS spectra of soybean lecithin and insulin in positive mode. (A) Soybean lecithin, a–d, expressed the main group peaks of soybean lecithin. (B)Insulin, the labelled number of charges is supposed to be insulin monomer. (�) Multiple charged ion peaks of Insulin.

multiple charged ion peaks appearing in ESI-MS spectrum(Fig. 3B). The three ion peaks of insulin were set as the stan-dard of screening insulin by ESI-MS.

To meet the requests of the determination of insulinby HPLC, liquid–liquid extracting method was applied toremove soybean lecithin and phenol from IES. Eight alcohols(n-butanol, n-pentanol, n-hexanol, n-heptanol, n-octanol,n-nonanol,n-decanol, andn-hendecanol) were selected to bethe extractant candidates because of the following reasons:the first was the solubility of phenol in alcohol is much morethan in water; the second was soybean lecithin could bedissolved in alcohols; the third was insulin could hardly bedissolved in alcohols; the fourth was the selected alcoholscould stratify from aqueous phase. One alcohol, which couldextract the most soybean lecithin and the least insulin, andmake the tailed peak of phenol disappear in the chromato-graphic spectrum, would be selected as the extractant for IES.The screening results by ESI-MS are shown inFigs. 4 and 5.The extracted effects of soybean lecithin in different alcoholsare exhibited inFig. 4, and the signal intensity sequenceof soybean lecithin in the alcohols was as follows:n-butanol >n-pentanol >n-hexanol >n-heptanol >n-octanol >n-nonanol >n-decanol >n-hendecanol.n-Butanol,n-pentanol, andn-hexanol showed considerable ability onextracting soybean lecithin.Fig. 5 shows that the quantityof insulin extracted byn-butanol was much more than othera antf ftere

d .2%,m andi em hro-

matographic spectrum of phenol (Fig. 6). The tailed peak ofphenol disappeared (Fig. 6A), and the peak area (126.1 inFig. 6A and 165.5 inFig. 6B) was reduced more than hun-dreds of times compared with that inFig. 2B (area: 11,076.4)and Fig. 2C (area: 11,104.4).Fig. 6B shows the area ofinsulin peak (area: 3021.6) was close to that inFig. 2A(3073.3), proving that the interference caused by phenol ininsulin measurement was eliminated byn-pentanol extrac-tion. The results above demonstrated thatn-pentanol shouldbe a suitable extractant for IES to meet the requests of insulinquantification.

F ed byd actantfo

lcohols, suggestingn-pentanol would be a better extractor IES if the tailed peak of phenol could disappear axtraction byn-pentanol.

To examine whether the tailed peak of phenol (Fig. 2B)isappeared after extraction, the phenol solution (0/m) and the solution containing phenol (0.2%, m/m)

nsulin (4 U/mL) were extracted byn-pentanol using the samethod, and then injected, respectively, to obtain the c

ig. 4. The overlapped ESI-MS spectra of soybean lecithin extractifferent alcohols in positive mode. A–H expressed the alcohols as extr

or ES, (A)n-butanol, (B)n-pentanol, (C)n-hexanol, (D)n-heptanol, (E)n-ctanol, (F)n-nonanol, (G)n-decanol, and (H)n-hendecanol.

Y. Luo et al. / Analytica Chimica Acta 553 (2005) 64–72 69

Fig. 5. The overlapped ESI-MS spectra of insulin extracted by different alco-hols in positive mode. A–H expressed the alcohols as extractant for insulinsolution, (A)n-butanol, (B)n-pentanol, (C)n-hexanol, (D)n-heptanol, (E)n-octanol, (F)n-nonanol, (G)n-decanol, and (H)n-hendecanol.

To validate the screening results by ESI-MS, we had usedn-butanol andn-hexanol to extract the insulin-loaded emul-sion system, but the results were not satisfied. We found thatthe test values of insulin content in IES extracted byn-butanolwere always lower than the calculated ones. The main rea-son was that water could dissolve inn-butanol at a certaindegree, so that a small amount of insulin could go into alco-hol phase with water. If extracted byn-hexanol (and otherhigher alcohols), the pressure of chromatographic columnsoon increased in a considerable speed because that too mucsoybean lecithin was left in the samples, leading to swiftlydecrease of column efficiency. These experimental results

were in accordance with that of the ESI-MS screening.n-Pentanol was found to be an optimized extractant for it couldeliminate soybean lecithin and phenol as much as possible,but most insulin was left. The ESI-MS screening results abovewere proved by HPLC results in return.

3.3. The optimized chromatographic conditions andvalidation of analytical method

The chromatographic conditions were optimized throughthe respects of eluent composition, eluting gradient, flow rate,column temperature, and detection wavelength. Sulphate orphosphate buffer system with acetonitrile was used as eluentfor the determination of insulin in previous studies[23–27],but the sulphate buffer with acetonitrile as eluent could notseparate phenol and insulin in IES effectively. On the con-trary, phosphate buffer solution (PBS) with acetonitrile aseluent exhibited good separation efficiency while the pHvalue of PBS was set from 3.0 to 3.5. The higher or lower pHvalue could result in the resolution between insulin and phe-nol decreasing greatly. The change of phosphate content hadonly a slight influence on the retention behavior of insulin andphenol, but more phosphate would increase the burden of theHPLC system, and less phosphate could not obtain enoughbuffering capacity. The eluting gradient and flow rate weres highr ntiont rom3 ud-i idea ateds sen-s

edw ining

F solutio ulina

ig. 6. The chromatographic spectra of insulin and phenol. (A) Phenolfter extraction byn-pentanol.

h

elected to obtain good chromatographic peak shape,esolution between insulin and phenol, and suited reteime of insulin. The column temperature could be set f0 to 40◦C according to insulin nature and previous st

es [23–27]. The characteristic UV absorptions of peptnd protein (214.4, 254.4, and 276.4 nm) were investigimultaneously with DAD in same scale, and the highestitivity of insulin was found at 214.4 nm.

The quantification of insulin by HPLC was performith external standard method, and all samples conta

n after extraction byn-pentanol and (B) the solution containing phenol and ins

70 Y. Luo et al. / Analytica Chimica Acta 553 (2005) 64–72

Fig. 7. The typical chromatographic spectra of ES and IES after extraction byn-pentanol. (A) ES and (B) IES.

Table 1Recovery results (n = 3)

Concentrationadded (U/mL)

Concentration founded(mean± S.D.) (U/mL)

Recovery (%)

3.20 (80%) 3.23± 0.01 100.94.00 (100%) 4.10± 0.01 102.54.80 (120%) 4.71± 0.01 98.1

soybean lecithin and phenol were extracted byn-pentanolbefore HPLC test. Under the optimized chromatographicconditions, insulin had a retention time of 8.784± 0.036 min(R.S.D.: 0.41%,n = 7), and phenol had a retention time of7.388± 0.016 min (R.S.D.: 0.22%,n = 7). The typical chro-matographic spectra of ES and IES after extraction byn-pentanol are shown inFig. 7. There was a clear resolution ofinsulin and phenol shown inFig. 7B (Rs = 5.92). Moreover,the specificity of the assay was clearly exhibited in Figs.2Aand7. The retention times of insulin were very close, andno interferences from the drug matrix (ES) in insulin detec-tion were observed (Fig. 7A). The theory plate number ofthe column for insulin detection was 81,712 m−1, and USP(Pharmacopoeia of United States) tailing of insulin peak was1.029.

The standard curves exhibited a good linearity betweenthe response (y) and the corresponding concentra-tion range of insulin (x) from 1.05 to 7.34 U/mL

Table 2The results of within-day precision (n = 6)

Time-sampling(min)

Concentrationfounded (U/mL)

Mean± S.D.(U/mL)

R.S.D.(%)

0 3.97

4.00± 0.02 0.5

75 4.00150 4.01233

(y = 17,113.944x + 49.665232). The correlation coefficient ofthe standard curve was calculated as 0.99936.

The recovery results are shown inTable 1. Mean recover-ies were obtained between 98.1 and 102.5%. The results ofwithin-day precision are presented inTable 2and the R.S.D.value was 0.5%. The LOD and LOQ of the present methodwere 0.01 and 0.03 U/mL, respectively.

3.4. The study of storage conditions for IES

To show the effects on storage conditions intuitively, wedefined degradation ratio (DR) as follows: the initial contentof insulin was set to basic value, and the DR of untreated IESshould be 0; then, the DR of the treated samples could becalculated as the following formula:

DR = CU − CT

CU× 100%

where DR is the degradation ratio;CU the average value ofinsulin concentration of untreated samples, which was testedthree times;CT is the average value of insulin concentrationof the treated samples, which was tested three times. Theexhibited DR below (expressed as mean± S.D.) was the lasttime point of different storage conditions).

With the analytical method, it was possible to analyze largea sultso ti rables % in1s ly,an get asbs be

25 4.0100 4.0075 4.01

mounts of samples rapidly and accurately. The test ref storage conditions are shown inFig. 8. The insulin conten

n IES placed under intense light decreased at considepeed, and the degradation ratio of insulin exceeded 200 days (DR = 26.1± 2.4%,n = 3, Fig. 8A). Insulin in IEStored at room temperature (25◦C) also decreased swiftnd the DR exceeded 15% in 1 month (DR = 17.3± 1.32%,= 3, Fig. 8C). Fig. 8B illustrates that the feasible stora

emperature for IES should be 4◦C, because the DR welow 15% (DR = 10.1± 1.5%, n = 3, Fig. 8B) after beingtored at 4◦C for 1 year. Therefore, insulin in IES would

Y. Luo et al. / Analytica Chimica Acta 553 (2005) 64–72 71

Fig. 8. Dependence of insulin biopotency on time under different storageconditions. (A) Placed under intense light (2500± 500 lx) for 10 days, (B)stored at 4◦C for 12 months, and (C) stored at 25◦C for 30 days. IES 1–9means nine samples with the same preparation method, and each curve meanone sample. Each point represents the DR value (expressed as mean± S.D.)of three tests.

easy to degrade under light or at room temperature but wasconsiderably stable at 4◦C in light proof condition.

The HPLC method with the pretreatment proceduredescribed here enables to measure insulin in IES accu-rately. Other methods proposed by previous studies wereable to detect insulin in different systems with credibleresults [23–27], but there were no intense interferencesfrom the drug matrix. However, both soybean lecithin andphenol in IES interfered in measuring insulin by HPLCintensely. ESI-MS was applied successfully to screen extrac-tants, and the results confirmed that the interferences fromsoybean lecithin and phenol were eliminated simultane-ously after extraction byn-pentanol. The validation exper-iments indicated that insulin in IES could be measured accu-rately and precisely with a suited pretreatment procedure byHPLC-UV.

4. Conclusion

n-Pentanol was selected by ESI-MS to be an appropriateextractant for IES. The interferences from the drug matrix(soybean lecithin and phenol) could be eliminated simul-taneously by usingn-pentanol as extractant. The validationexperiments indicated that the HPLC method with extractionb PLCm ngt IESsd

A

ch-n orsw ityo

R

E.91.er-

71.995)

4)

133.863.

[[[ 9.

s

y n-pentanol was a sensitive, reliable, and specific Hethod for identifying or quantifying insulin in IES. Usi

he method above, the study of storage conditions forhowed that IES would be stable at 4◦C in light proof con-ition.

cknowledgements

This study was supported by China National Key Teologies R&D Program (2003BA310A12). The authould like to thank Dr. Xiqun Sheng (Huazhong Universf Science & Technology) for helpful discussions.

eferences

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