Mass Spectrometric Identification of Degradation Products of Insulin

and unambiguous evidence for an administration of this insulin analogue. ... positions B31 and B32 (C-terminus, Figure 1b). These modifica-. * Cor...
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Anal. Chem. 2007, 79, 2518-2524

Mass Spectrometric Identification of Degradation Products of Insulin and Its Long-Acting Analogues in Human Urine for Doping Control Purposes Andreas Thomas,† Mario Thevis,*,† Philippe Delahaut,‡ Alain Bosseloir,§ and Wilhelm Scha 1 nzer†

Center for Preventive Doping Research and Institute of Biochemistry, German Sport University Cologne, Carl-Diem Weg 6, 50933 Cologne, Germany, Laboratory of Hormonology, Centre d’Economie Rurale, Rue du Point du Jour 8, 6900 Marloie, Belgium, and ZenTech, Parc Scientifique du Sart-Tilman, Avenue du Pre` -Aily 10, 4031 Angleur, Belgium

The search for target analytes to uncover the misuse of long acting insulin analogues (Lantus, Insulin Glargine; Levemir, Insulin Detemir) in doping control samples led to the identification of several degradation products of insulin or its synthetic analogues. Specimens obtained from healthy volunteers or patients and athletes suffering from diabetes mellitus contained DesB30, DesB24-30, and DesB25-30 human insulin or DesB30-32, DesB31-32, and DesB24-32 Lantus, respectively. Analytes were purified from urine by immunoaffinity chromatography (IAC) with subsequent liquid chromatographytandem mass spectrometry analysis. The employed analytical procedure was validated for qualitative determination considering the main metabolic products DesB30 human insulin and DesB30-32 Lantus. The occurrence of the identified Lantus degradation products in urine provided the direct and unambiguous evidence for an administration of this insulin analogue. For the determination of surreptitious Levemir or recombinant human insulin applications, an unequivocal argument was not detected, but promising approaches based on a modified insulin degradation profile with altered relative intensities of metabolites are presented. Long- and intermediate-acting insulin analogues are produced recombinantly to obtain improved pharmacokinetic injection-toonset profiles for the treatment of diabetes mellitus.1 While rapid acting insulin formulations have been applied postprandial as bolus insulins, long acting analogues have ensured the steady supplement of basal insulin plasma levels. Commercially available insulin formulations with a prolonged action profile are Lantus (Insulin Glargine, LAN), Levemir (Insulin Detemir, LEV), and recombinant human insulin (HI) with additions (e.g., NPH, neutral protamine hagedorn) delaying the bioavailability of the active hormone in the systemic circulation.

Figure 1. Structure images of (a) HI, (b) LAN, and (c) LEV. Modifications are marked in gray, and identified cleavage sites are pointed with arrows.

* Corresponding author: Tel.: 0049221-4982-7070. Fax: 0049221-497-3236. E-mail: [email protected]. † German Sport University Cologne. ‡ Centre d’Economie Rurale. § ZenTech. (1) Hermansen, K.; Fontaine, P.; Kukolja, K. K.; Peterkova, V.; Leth, G.; Gall, M. A. Diabetologia 2004, 47, 622-629.

LAN is a synthetic insulin analogue that differs in its amino acid sequence from HI (Figure 1a) by exchanging the amino acid residue asparagine to glycine at position 21 of the A-chain, and the B-chain is prolonged by two additional arginine residues at positions B31 and B32 (C-terminus, Figure 1b). These modifica-

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10.1021/ac062037t CCC: $37.00

© 2007 American Chemical Society Published on Web 02/15/2007

tions resulted in an elevated isoelectric point and, thus, through a decreased solubility at physiological pH values, microprecipitation after subcutaneous injection.2-4 LEV [LysB29-(N-tetradecanoyl) Des(B30) human insulin], a synthetic analogue with protracted bioavailability, possesses a combined increase of self-association to hexameric aggregates and albumin binding due to acylation of the amino acid residue B29lysine with myristic acid (Figure 1c). The bioactivity of LEV is decreased compared to HI, which requires the application of approximately the 4-fold amount compared to HI formulations.1,5,6 NPH insulins are intermediate or long acting recombinant HI preparations. The pharmacokinetic behavior is attributable to reversible protamine binding in subcutaneous tissue and, thus, to a delayed release into the systemic circulation.7,8 Determination of long acting insulin analogues in urine is of utmost interest for doping control purposes and pharmacokinetic or clinical sciences. Unfortunately, intact LAN or LEV is not observed in urine specimens employing LC-MS/MS-based approaches according to earlier studies with rapid-acting analogues.9,10 Liver and kidney are generally known for owning main metabolic pathways and removal from systemic circulation of insulin.11 The adsorption and degradation in glomerula and postglomerula of the kidney is highly efficient, and less than 1% of the intact hormone is excreted into the urine.12,13 Several degradation products were identified using 125I-labeled insulin after incubation with hepatocytes or other insulin-sensitive tissues and subsequent HPLC analyses.14,15 Another recent study, designed to investigate the pharmacokinetic behavior of LAN after subcutaneous injection, demonstrated that insulin metabolism starts at the injection site.4 Products of these degradation processes consist of structures with a truncated A- or B-chain with unaffected disulfide bonds,11,12 but the presence of these metabolites in human urine has not been established so far. The misuse of insulin and its synthetic analogues in elite and amateur sports has been reported earlier,16,17 although its performance-enhancing potential is still under discussion.18-21 The aim (2) Owens, D. R.; Tinbergen, J. P.; Coates, P. A.; Kurzhals, R.; Luzio, S. D. Diabetes Care 2000, 23, 813-819. (3) Rosenstock, J.; Park, G.; Zimmermann J. Diabetes Care 2000, 23, 11371142. (4) Kuerzel, G. U.; Shukla, U.; Scholtz, E. H.; Pretorius, S. G.; Wessels, D. H.; Venter, C.; Potgier, M. A.; Lang, A. M.; Loose, T.; Brenhardt, E. Curr. Med. Res. Opin. 2003, 19, 34-40. (5) Heise, T.; Nosek, L.; Biilmann Ronn, B.; Endahl, L.; Heinemann, L.; Kapitza, C.; Draeger, E. Diabetes 2004, 53, 1614-1620. (6) Danne, T.; von Schuetz, W.; Lu ¨ pke, K.; Gall, M. A.; Walte, K. Diabetes Care 2003, 26, 3087-3092. (7) Scholtz, H. E.; Pretorius, S. G.; Wessels, D. H.; Becker, R. H. A. Diabetologia, 2005, 48, 1988-1995. (8) Hoeg-Jensen, T.; Havelund, S.; Nielsen, P.; Markussen, J. J. Am. Chem. Soc. 2005, 127, 6158-6159. (9) Thevis, M.; Thomas, A.; Delahaut, P.; Bosseloir, A.; Schaenzer, W. Anal. Chem. 2005, 77, 3579-3585. (10) Thevis, M.; Thomas, A.; Delahaut, P.; Bosseloir, A.; Schaenzer, W. Anal. Chem. 2006, 78, 1897-1903. (11) Duckworth, W. C.; Bennet, R. G.; Hamel, F. G. Endocr. Rev. 1998, 19, 608624. (12) Duckworth, W. C.; Hamel, F. G.; Liepnieks, J.; Peavy, D.; Frank, B.; Rabkin, R. Am. J. Physiol.: Endocrinol. Metab. 19 1989, 256, E208-E214. (13) Fawcett, J.; Rabkin, R. Endocrinology 1995, 136, 39-45. (14) Benzi, L.; Cecchetti, P.; Ciccarone, A. M.; Di Cianni, G.; Iozzi, L. C.; Caricato, F.; Navalesi, R. J. Chromatog. 1990, 534, 37-46. (15) Seabright, P. J.; Smith, G. D. Biochem. J. 1996, 320, 947-956. (16) Sonksen, P. H. J. Endocrinol. 2001, 170, 13-25. (17) Dawson, R. T.; Harrison, M. W. Br. J. Sport Med. 1997, 31, 259.

of the present study was the development of an analytical procedure enabling the determination of an application of long acting insulin formulations in regular urinary doping control specimens. Metabolic degradation products were identified and employed as target analytes for qualitative determination utilizing liquid chromatography-tandem mass spectrometry after highly specific IAC sample preparation. EXPERIMENTAL SECTION Materials and Chemicals. OASIS HLB solid-phase extraction cartridges (60 mg, 3 ccm) were obtained from Waters (Milford, MA), and acetonitrile (HPLC grade), trisodium phosphate dodecahydrate (p.a.), sodium chloride (p.a.), and acetic acid (glacial) were purchased from Merck (Darmstadt, Germany). Trifluoracetic acid (99+%), tris(carboxyethyl)phosphine hydrochloride (TCEP-HCl), endoproteinase Lys-C from Lysobacter enzymogenes, and bovine insulin were from Sigma (St. Louis, MO). Lantus (Insulin Glargine), Levemir (Insulin Detemir), and recombinant human insulin were supplied by Novo Nordisk (Princeton, NJ), Aventis (Kansas City, MO), and Aventis (Frankfurt, Germany), respectively. DesB30 human insulin was kindly provided by Dr. T. Hoeg-Jensen from Novo Nordisk (Bagsvaerd, Denmark). The anti-insulin immunoaffinity gel (0.5 mL/IAC, 10 mg IgG/mL) was obtained from CER (Marloie, Belgium). Urine Samples. All experiments and validation steps were performed with fortified urine samples obtained from healthy male and female volunteers. Furthermore, urine samples were analyzed from athletes or patients, suffering from diabetes mellitus and declaring the continual regimen with recombinant HI, LAN, or LEV. Hydrolysis of LAN. Enzymatic hydrolysis of LAN was performed using endoproteinase Lys-C from Lysobacter enzymogenes to obtain DesB30-32 LAN reference compound. To 1 mL of a solution containing 10 pmol/µL of LAN in 100 mM ammonium bicarbonate buffer (pH 7.5) was added 20 µL of reconstituted enzyme solution (4.8 units/mL), and the mixture was incubated at 37 °C for 2 h. Hydrolysis was stopped by adding 20 µL of glacial acetic acid. Stock and Working Solutions. A solution containing 10 pmol/µL of bovine insulin in 2% acetic acid was used as internal standard stock solution. DesB30 HI and DesB30-32 LAN stock solutions contained 10 pmol/µL in 2% acetic acid and were freshly diluted before use to a final concentration of 0.01 pmol/µL in 2% acetic acid. These working standard solutions also contained a 10-fold carrier-excess of bovine insulin (0.1 pmol/µL), which was added prior to the target analytes in order to saturate active surfaces. All dilution steps were performed in polypropylene tubes, and stock solutions, stored at 2-8 °C, were found to be stable for 1 month. Mass Spectrometry. All reference spectra were measured on an Applied Biosystems Qtrap 4000 mass spectrometer (Foster (18) Ebeling, P.; Bourey, R.; Koranyi, L.; Tuominen, J. A.; Groop, L. C.; Hendriksson, J.; Mueckler, M. Sovijarvi, A.; Koivisto, V. A. J. Clin. Invest. 1993, 92, 1623-1631. (19) Laurent, D.; Hundal, R. S.; Dresner, A.; Price, T. B.; Vogel, S. M.; Falk Petersen, K.; Shulmann, G. I. Am. J. Physiol.: Endocrinol. Metab. 2000, 278, E663-E668. (20) Rodnik, K. J.; Haskell, W. L.; Swislocki, A. L. M.; Foley, J. E.; Reaven, G. M. Am. J. Physiol.: Endocrinol. Metab. 16 1987, 253, E489-E495. (21) Takala, T. O.; Nuutila, P.; Knuuti, J.; Luotolathi, M.; Yki-Jarvinen, H. Am. J. Physiol.: Endocrinol. Metab. 39 1999, 276, E706-E711.

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Table 1. Selected Mass Spectrometric Parameters and Validation Results for HI, LEV, and LAN Degradation Products

analytes

MW [Da]

precursor ion, m/z

collision offset voltage [V]

recovery [%]

LOD (S/N > 3) [pg/mL]

precision at LOD [%}

R

calcd curve (25-200 pg/mL) intercept slope approximation

DesB30 human insulin DesB24-30 human insulin DesB25-30 human insulin

5706.6 4922.6 5069.8

1142.3 1231.7 1268.5

75 75 75

HI and LEV 109 n/a n/a

25 n/a n/a

12 n/a n/a

0.98 n/a n/a

0.242 n/a n/a

0.006 n/a n/a

linear n/a n/a

DesB30-32 Lantus DesB31-32 Lantus DesB24-32 Lantus

5649.5 5750.6 4865.6

1130.9 1151.1 1217.4

75 75 75

LAN 97 n/a n/a

25 n/a n/a

14 n/a n/a

0.995 n/a n/a

0.075 n/a n/a

0.024 n/a n/a

linear n/a n/a

City, CA) using an electrospray ion source in positive mode. Selected mass spectrometric parameters are summarized in Table 1. Identity and purity of the utilized reference compounds DesB30 HI and DesB30-32 LAN were proven by measurements using a 1100 Series capillary liquid chromatograph from Agilent (Palo Alto, CA) coupled to an LTQ-Orbitrap mass spectrometer from Thermo (Bremen, Germany). Deconvolution of ESI-full scan spectra confirmed the accurate molecular masses of 5706.6 Da for DesB30 HI and 5649.5 Da for DesB30-32 LAN. Sample Preparation. Sample preparation was adapted from a formerly developed procedure and is described in detail elsewhere.9,10 Briefly, 25 mL of urine was fortified with the internal standard, acidified, and vortex mixed. After OASIS solid-phase extraction (SPE), the cartridge was eluted directly onto the IAC. The IAC sample mixture was incubated for 30 min at room temperature, and target analytes were eluted directly onto another SPE. Concentrated extracts were evaporated to dryness and analyzed using LC-MS/MS. In a complementary analysis, intramolecular disulfide bonds were cleaved by adding TCEP-HCL to the reconstituted sample solution, and the reduced sample was also injected into the LCMS/MS system. LC-MS/MS. LC was performed on an Agilent 1100 Series high performance liquid chromatograph (Palo Alto, CA) coupled to an Applied Biosystem Qtrap 4000 mass spectrometer (Foster City, CA). The LC was equipped with a Zorbax StableBond guard column (1 mm × 17 mm, 5 µm particle size) and a Zorbax 300SBC18 analytical column (1 × 50 mm, 5 µm particle size, 300 Å pore size) with an ambient column oven temperature of 40 °C. The mobile phases consisted of 0.1% acetic acid with 0.01% TFA (phase A) and a mixture of 0.1% acetic acid with 0.01% TFA and acetonitrile (2:8, v:v) (phase B). The gradient started at 72% A, ending at 35% A after 15 min with a flow rate of 70 µL/min. Subsequently a 25 min equilibration period was added. The mass spectrometer was operated in positive ion spray mode with a needle voltage of 5500 V. Parameters such as declustering potential, ion trap fill time, and entry barrier were optimized for isolation and detection of the 5-fold protonated molecules of HI, DesB30 HI, DesB30-32 LAN, and DesB31-32 LAN. Product ion spectra were measured at collision energies of 75 eV utilizing nitrogen as collision gas (6.0 × 10-3 Pa). Validation Parameter. All validation steps were affected by the lack of available reference compounds and, thus, were focused on DesB30 HI and DesB30-32 LAN, only. Specificity was shown 2520 Analytical Chemistry, Vol. 79, No. 6, March 15, 2007

by preparing 10 different urine samples as described above. The limit of detection (LOD) was determined by comparison of the signal-to-noise ratio of urine samples, fortified with 25 pg/mL of both analytes and blank urine samples measured for specificity. Considering a signal-to-noise ratio g3, the relative standard deviations for a 6-fold determination of each analyte at a concentration level of 25 pg/mL were calculated. In order to determine the recovery of the preparation procedure, six urine samples were spiked at a concentration of 200 pg/mL and prepared as described above. The obtained results were compared to six sample preparations, spiked with 200 pg/mL prior to the evaporation in the vacuum centrifuge. Recovery rates were calculated by comparison using ratios of the target peak areas to the internal standard of both series. Another set of urine samples was fortified with 25, 50, 75, 100, 125, 150, and 200 pg/mL of each reference compound and measured once to demonstrate the linearity of the signal ratios in this concentration range. The stability of the analytes in urine was proven by analyses of a fortified sample before and after 2, 4, and 8 weeks of storage time at 4 °C and -20 °C. RESULTS AND DISCUSSION Characterization of Metabolic Products. The absence of intact synthetic insulin molecules in urine samples obtained from athletes or patients suffering from diabetes mellitus and the availability of therapies based on LAN or LEV prompted the investigation of possible metabolites or degradation products occurring in the respective urine samples. Metabolic pathways of insulin in liver and kidney have been described in literature for over 30 years,22 and hepatic degradation and renal clearance of insulin by degrading enzymes or glomerula filtration is a well examined phenomenon.11 After in vitro incubation of 125I-labeled insulin analogues with isolated hepatocytes and treatment with insulin degrading enzyme or insulin protease from human fibroblasts, several cleavage sites at different A- and B-chain positions (e.g., A13/14, A14/15, B9/10, B10/11, B13/14, B16/ 17, B24/25, and B25/26)11,12,14,15,23,24 with intact disulfide bonds and presence of partially degraded extracellular insulin11 were well established. However, none of these products were discovered in urine samples obtained from patients treated with different long (22) Katz, A. I.; Rubenstein, A. H. J. Clin. Invest. 1973, 53, 1113-1121. (23) Authier, F.; Danielsen, G. M.; Kouach, M.; Briand, G.; Chauvet, G. Endocrinology 2001, 142, 276-289. (24) Stentz, F. B.; Kitabchi, A. E.; Schilling, J. W.; Schronk, L. R.; Seyer, J. M. J. Biol. Chem. 1989, 264, 20275-20282.

Figure 2. ESI-product ion chromatograms of a urine sample from a patient treated with LEV representing (a) the 5-fold protonated precursor ion at m/z 1142.3 for DesB30 HI, (b) 4-fold protonated precursor ion at m/z 1231.7 for DesB24-30 HI, and (c) 4-fold protonated precursor ion at m/z 1268.5 for DesB25-30 HI. Respective product ion spectra are shown with characteristic amino acid sequence tags.

acting insulin formulations except for DesB24-30 HI. By extended analyses, unknown insulin degradation products with cleavage sites at positions B23 and B29 were identified as illustrated in Figure 1 (arrows). ESI-product ion chromatograms with corresponding MS/MS spectra of the 4- or 5-fold protonated molecules determined in a urine sample obtained from a patient administering LEV are shown in Figure 2. DesB30 HI was identified with a retention time of 17.5 min by isolating the 5-fold protonated precursor ion at m/z 1142.3, which yielded the characteristic B-chain-derived product ions (B)y2, (B)y3, and (B)y4 at m/z 244, 345, and 508, respectively. For DesB24-30 HI and DesB25-30 HI, distinct signals at 16.4 and 17.1 min were obtained after fragmentation of the 4-fold protonated precursors at m/z 1231.7 and 1268.5. Calculated molecular weights plus diagnostic product ions (B)y3 and (B)y4 at m/z 361, 379 and 418, 508 enabled their identification as HI degradation products. A urine sample obtained from a patient treated with LAN was prepared and measured as described above. ESI-product ion chromatograms with corresponding mass spectra (Figure 3) allowed the identification of DesB30-32, DesB31-32, and DesB2532 LAN in this sample with signals at 17.2, 16.8, and 16.3 min, respectively. Due to identical amino acid sequences of truncated B-chains of LAN, HI, and LEV, corresponding diagnostic y2, y3, and y4 product ions were employed for identification of DesB3032 LAN and DesB25-32 LAN derived from distinct precursor ions at m/z 1130.9 (M + 5H)5+ and 1217.4 (M + 4H)4+. DesB31-32 LAN, that is attributed to the lack of two arginine residues at the C-terminus of the B-chain, was characterized by fragment ions (B)y3-y1, (B)y3, and (B)y4 at m/z 226, 345, and 446, in accordance to collision-induced dissociation of intact HI10 (not shown), with

a considerably abundant 5-fold protonated precursor ion at m/z 1151.1. In order to substantiate the proposed primary sequence of insulin degradation products and confirm the suggested cleavage sites, measurements of truncated B-chains only were performed after reduction of the disulfide bonds with TCEP-HCl solution. An ESI-product ion chromatogram with corresponding mass spectra of the 3-fold protonated molecule at m/z 849.2 of Des2430 B-chain of HI is shown in Figure 4. The urine sample was obtained from a diabetic athlete, who declared the administration of recombinant HI. The abundant signal at 16.6 min yielded a comprehensive amino acid sequence tag information representing the y- and b-ion series from y3 to y8 and b102+ to b162+, which verified the identity of DesB24-30 HI. In vivo insulin degradation has been described as a timedependent process,15,24 and long acting insulin formulations are subjected to a protracted exposure to metabolic processes due to a prolonged half-life in subcutaneous tissue or systemic circulation. Thus, in urine samples provided by patients treated with LAN or LEV, intact insulin analogues with complete amino acid sequence were not present in detectable amounts. Until today no attempt has been made to determine possible insulin metabolites in human urine due to assumed complete clearance from circulation.11,12 Commonly used immunochemical insulin assays were, with respect to their specific epitope, unable to distinguish between the intact insulin sequence or its slightly different metabolites.25 Furthermore, DesB30 HI, obviously derived as metabolite from HI (endogenous and recombinant), was (25) Owen, W. E.; Roberts, W. L. Clin. Chem. 2004, 50, 257-259.

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Figure 3. ESI-product ion chromatograms of a urine sample from a patient treated with LAN representing (a) the 5-fold protonated precursor ion at m/z 1130.9 for DesB30-32 LAN, (b) 5-fold protonated precursor ion at m/z 1151.1 for DesB31-32 LAN, and (c) 4-fold protonated precursor ion at m/z 1217.4 for DesB25-32 LAN. Respective product ion spectra are shown with characteristic amino acid sequence tags.

Figure 4. ESI-product ion chromatogram (a) of a urine sample from an elite athlete, treated with recombinant HI after reduction of the intramolecular disulfide bonds with TCEP-HCl solution. The 3-fold protonated precursor ion at m/z 849.2 yielded in a product ion spectrum (b) providing a comprehensive amino acid sequence tag of DesB25-30 HI-B-chain.

not mentioned in the literature until today due to omitted analytical findings after incubation of insulin with hepatocytes, kidney cells, or other insulin sensitive tissues.11,12,14,15,23,24 Doping Control Aspects. The identification of LAN metabolites (including DesB24-32, DesB30-32, and DesB31-32) in 2522 Analytical Chemistry, Vol. 79, No. 6, March 15, 2007

urine samples provides direct and unambiguous evidence for usage of the insulin analogue as the A-chain of unmodified LAN (see Figure 1), ensuring the distinction from HI degradation products by their attenuated molecular weights. Those metabolites are distinct target analytes to determine the misuse of this long

Figure 5. DesB30 HI to HI ratios in urine samples from 13 healthy volunteers with endogenously produced HI, only (black), three diabetic patients treated with recombinant HI (gray), and one sample from a patient after LEV application (white).

acting insulin analogue in elite sport doping controls. The immobilized antibodies utilized for IAC possess an epitope that is located at the N-terminus of the A-chain, so all target analytes including intact HI, LEV, and LAN as well as metabolic products were purified simultaneously. In contrast to the unambiguous identification of LAN misuse by means of diagnostic metabolites, a mass spectrometric differentiation between DesB30 HI originating from HI or LEV is missing. Trace amounts of degradation products resulting from a C-terminal truncated B-chain were frequently observed in urine samples obtained from healthy, nondiabetic volunteers without exogenous insulin treatment within this study. So, unfortunately, the presence and determination of those degradation products supply an ineligible evidence for a LEV application. However, the analyses of urine samples from patients treated with LEV or recombinant HI revealed a considerably elevated DesB30 HI to HI ratio compared to samples from healthy volunteers. In the case of LEV this phenomenon might be explained by a prolonged metabolic exposure as well as a decreased biological activity and the accordingly increased amount of administration.26,27 In Figure 5, the DesB30 HI to HI ratios of 17 urine samples are presented. Thirteen samples were obtained from healthy volunteers with endogenously produced HI only (black), three samples were provided by patients treated with recombinant HI (gray), and one sample was supplied by a patient after LEV application (white). The diagram illustrates the increase of DesB30 HI to HI ratios in patients treated with exogenous insulin. Generally, a doping control sample is considered to contain a prohibited substance when a relevant ratio exceeds a value normally found in humans and which is unlikely to be consistent with normal endogenous production.28 So metabolic profiling of insulin degradation products in urine from healthy volunteers compared to profiles from volunteer/patients treated with exogenous insulin potentially (26) Havelund, S.; Plum, A.; Ribel, U.; Jonassen, I.; Volund, A.; Marussen, J.; Kurtzhals, P. Pharm. Res. 2004, 21, 1498-1504. (27) Kurtzhals, P.; Schaeffer, L.; Sorensen, A.; Kristensen, C.; Jonassen, I.; Schmid, C.; Trueb, T. Diabetes 2000, 49, 999-1005. (28) World Anti-Doping Agency. The 2007 Prohibited List, http:// www.wada-ama.org/rtecontent/document/2007_List_En.pdf; Oct 20, 2006.

provides a promising, though yet unvalidated, approach to uncover the misuse of recombinant HI or LEV for doping control. Validation Results. A doping control procedure was validated for the metabolic products DesB30 HI and DesB30-32 LAN for qualitative purposes including the parameters of recovery, limit of detection (LOD), linearity, precision at the LOD, specificity, and stability. All validation parameter were calculated regarding the peak areas resulting from total ion current of the respective product ion chromatogram. Main results are summarized in Table 1. Recoveries of 96% and 109% for DesB30 HI and DesB30-32 LAN at a concentration level of 200 pg/mL with a precision e15% (n ) 6 + 6) were obtained for both analytes. Utilizing the chromatographic background noise of 10 blank urine samples at the corresponding retention time, a limit of detection of approximately 25 pg/mL was estimated with a S/N > 3. Calibration curves were prepared between 25 and 200 pg/mL. The DesB30 HI calibration curve was slightly elevated due to a concentration of endogenous DesB30 HI in the blank urine of about 40 pg/mL. Peak area ratios of the product ions normalized to the internal standard (bovine insulin) were used for the evaluation of linearity, and according to Mandel,29 linear approximation is permitted. Precision of less than 15% (n ) 6) at 25 pg/mL (LOD) was achieved for both compounds, and specificity was shown by analysis of 10 different urine samples with no interfering signals in the product ion scan chromatograms at the expected retention times for DesB30-32 LAN and DesB31-32 LAN. The presence of DesB30 HI strongly correlates to the amount of HI in the respective urine, and none of the analyzed samples were entirely free of target compound (DesB30 HI). Analyses of fortified urine samples stored at 4 °C and -20 °C showed no degradation of the target analytes after 2, 4, and 8 weeks of storage time. CONCLUSION The developed and validated procedure provides a fast and reliable way to elucidate the potential misuse of the long acting insulin analogue LAN in regular urinary doping control specimens. Within this study DesB24-32, DesB30-32, and DesB31-32 LAN were presented as target analytes and the incorporation of these degradation products in existing analytical procedures for rapid acting insulin analogues is possible and enhances its effectiveness.9,10 Unambiguous evidence for determination of prohibited LEV or recombinant HI administration in nondiabetic athletes was not demonstrated definitely, but indications for different metabolic processes resulting in a modified hormone excretion profile were observed. Future studies comparing normal urinary DesB30 HI to HI ratios will provide information whether a profile might serve as indication for insulin misuse. The identified insulin degradation products DesB24-30 HI and DesB30 HI possess nearly full biological activity4,30-32 and thus provide, in addition to doping control purposes, interesting new approaches concerning pharmacokinetic, clinical, or forensic sciences. All chemicals and materials employed in this study are commercially available or (29) Mandel, J. The Statistical Analyses of Experimental Data; John Wiley & Sons: New York, 1964. (30) Chang, S. G.; Choi, K. D.; Jang, S. H.; Shin, H. C. Mol. Cell 2003, 16, 323330. (31) Yang, S. Z.; Huang, Y. D.; Jie, X. F.; Feng Y. M.; Niu J. Y. World J. Gastroenterol. 2000, 6 (3), 371-373. (32) Kruse, V.; Jensen, I.; Permin, L.; Heding, A. Am. J. Phisiol.: Endocrinol. Metab. 35 1997, 272, E1089-E1098.

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simple laboratory synthesis (hydrolysis of LAN) is possible, which allows fast method transfer to other laboratories.

Novo Nordisk (Bagsvaerd, Denmark) for providing DesB30 HI reference compound.

ACKNOWLEDGMENT This project has been carried out with support from the WorldAnti-Doping Agency and the Manfred-Donike-Institute for Doping Analysis. The authors are grateful to Dr. T. Hoeg-Jensen from

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Received for review October 31, 2006. Accepted January 19, 2007. AC062037T