Selective Analysis of Secondary Amino Acids in Gelatin Using Pulsed

Aug 8, 2007 - An extraction scheme was implemented prior to HPLC-IPAD analysis ... to chromatographic analysis allowed the use of isocratic mobile-pha...
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Anal. Chem. 2007, 79, 6615-6621

Selective Analysis of Secondary Amino Acids in Gelatin Using Pulsed Electrochemical Detection Jason D. Russell,†,‡ John M. Dolphin,‡ and Miles D. Koppang*,†

Department of Chemistry, University of South Dakota, Vermillion, South Dakota, 57069, and GELITA USA, Inc., Sioux City, Iowa 51106

A method was developed for selective analysis of the secondary amino acids proline and 4-hydroxyproline from gelatin hydrolysates using anion-exchange high-performance liquid chromatography followed by integrated pulsed amperometric detection (HPLC-IPAD). An extraction scheme was implemented prior to HPLC-IPAD analysis to isolate the secondary amino acids by the removal of primary amino acids through derivatization with o-phthalaldehyde followed by solid-phase extraction with C18 packed columns. The use of the IPAD technique eliminated the need for a second derivatization step to detect secondary amino acids. The removal of interfering primary amino acids prior to chromatographic analysis allowed the use of isocratic mobile-phase conditions to achieve effective and efficient separation of the amino acids. This led to a more precise and accurate quantitation of their content in gelatin hydrolysates. Detection limits approach 10 parts per billion (∼2 pmol/injection) with a chromatographic analysis time under 8 min. The ratios of secondary amino acids, in addition to their abundances, were used to distinguish gelatin manufactured from bovine, porcine, and fish raw material sources. Precise knowledge of amino acid composition is essential for an unambiguous identification of a protein or peptide. In addition to identification, elucidation of a protein’s structure and function is greatly aided by precise knowledge of the specific types and abundances of amino acids present. For industries that produce edible or pharmaceutical products, detection and quantitation of amino acids may also be an important quality control feature ensuring product safety and manufacturing efficiency. The analysis of amino acid mixtures is hampered by the absence of a suitable chromophore on the majority of the naturally occurring amino acids. A common technique used to detect aliphatic amines and amino acids involves derivatization (tagging) of the analyte with a reagent specific for primary or secondary amines. The tagged amine or amino acid is then chromophoric, fluorophoric, or electrochemically active, depending on the nature of the tag. High-performance liquid chromatography (HPLC) is routinely used in conjunction with a large variety of tagging reagents and pre- or postcolumn derivatization techniques to * To whom correspondence should be addressed. E-mail: mkoppang@ usd.edu. † University of South Dakota. ‡ GELITA USA, Inc. 10.1021/ac070819w CCC: $37.00 Published on Web 08/08/2007

© 2007 American Chemical Society

separate and detect amino acids.1,2 Tagging amino acids, followed by spectroscopic or electrochemical detection, offers a less expensive option than techniques that require mass analyzers such as high-performance liquid chromatography mass spectrometry (HPLC-MS) and capillary electrophoresis mass spectrometry. The convenience of modular chromatographic equipment gives a small laboratory the flexibility to perform several types of analyses (electrochemical, conductivity, UV-vis, fluorescence) using interchangeable components without investing a significant sum of money on dedicated instrumentation. An alternative approach to amino acid tagging takes advantage of anodic electrochemical detection of amino acids at noble metal electrodes using multistep potential-time waveforms generally referred to as pulsed electrochemical detection. This technique, first developed by Johnson and co-workers,3 uses repeating multistep potential-time waveforms incorporating an anodic detection step followed by oxidative cleaning and cathodic reactivation of the noble metal electrode surface. The pulsed nature of the technique greatly improved electrode stability by minimizing electrode fouling commonly encountered in constant potential (dc) applications. The multistep potential-time waveform became known as pulsed amperometric detection (PAD) and allowed for the electrochemical detection of amino acids, catalyzed by the surface oxide formation at the electrode surface (mode II detection) without pre or postcolumn derivatization.4 A consequence of the mode II detection scheme is the production of a large baseline current. The baseline current has a tendency to drift toward anodic values, resulting from surface oxide formation and dissolution at the electrode surface. The addition of a rapid cyclic scan during the detection potential of the waveform, followed by the integration of the resulting current, greatly reduced the problems with the baseline. This technique, formerly known as potential-sweep pulsed coulometric detection,5-7 is now commonly called integrated pulsed amperometric detection (IP(1) Li, F.; Lim, C. K. In Handbook of Derivatives for Chromatography, 2nd ed.; Blau, K., Halket, J. M., Eds.; John Wiley & Sons Ltd.: Chichester, England, 1993; pp 158-164. (2) Lunn, G.; Hellwig, L. C. Handbook of Derivatization Reactions for HPLC; John Wiley & Sons, Inc.: New York, 1998; pp 625-820. (3) Polta, J. A.; Johnson, D. C. J. Liquid Chromatogr. 1983, 6, 1727-1743. (4) Johnson, D. C.; LaCourse, W. R. Anal. Chem. 1990, 62, 589A-597A. (5) Welch, L. E.; LaCourse, W. R.; Mead, D. A., Jr.; Johnson, D. C. Anal. Chem. 1989, 61, 555-559. (6) LaCourse, W. R. Pulsed Electrochemical Detection in High-Performance Liquid Chromatography; John Wiley & Sons, Inc.: New York, 1997. (7) Neuburger, G. G.; Johnson, D. C. Anal. Chem. 1988, 60, 2288-2293. (8) Johnson, D. C.; LaCourse, W. R. Electroanalysis 1992, 4, 367-380.

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AD).4,6 Separation by an anion-exchange LC column has been shown to be amenable to pulsed amperometric detection of amino acids resulting in a relatively selective, sensitive assay.4,5,7,9 HPLCIPAD continues to be a popular technique for the separation and detection of amino acids and biogenic amines.10-15 Gelatin is a protein extracted from collagen-containing materials, most commonly from bovine bones and hide and porcine skin. Commercial production of gelatin from collagenous materials typically begins with treatment of the raw material with aqueous acid or base that serves to swell the collagen in preparation for a thermal extraction of gelatin.16 Of the more common pretreatment processes, type A refers to an acid pretreatment while type B refers to an alkaline process. Gelatin has traditionally been used in applications ranging from pharmaceutical capsules and photographic films to confectionary foodstuffs and adhesives. However, the use of gelatin and its enzymatically produced hydrolysates have become seemingly ubiquitous with their use extending far beyond the traditional realms into areas such as cosmetics, microencapsulation,17 and tissue modeling for ballistics applications.18 With such a wide variety of uses, the full characterization of a gelatin has gained increased importance in order to optimize its use in any particular process or application. A unique property of animal-derived collagenous protein is the abundance of the secondary amino acid, 4-hydroxyproline, the structure of which aids in stabilizing the triple-helix structure of collagen.19 Generally, greater than 12% of the amino acid content, by dry protein weight, is in the form of 4-hydroxyproline. Together with the amino acid proline, these secondary amino acids comprise nearly 25% of the amino acid weight in dry, ash-free gelatin extracted from mammalian species.20 The detection of 4-hydroxyproline has long been used to estimate the amount, or presence, of collagenous protein (gelatin) in a sample of animal origin.21 The amounts of secondary amino acids found in collagenous proteins tends to differ from species to species. In warm-blooded animals, the amount of 4-hydroxyproline in collagen is closely related to the body temperature. In cold-blooded animals, the amount of 4-hydroxyproline in collagen is closely related to the environmental temperature.22 Precise quantitation of the amounts of secondary amino acids in gelatins may prove to be a useful (9) Clarke, A. P.; Jandik, P.; Rocklin, R. D.; Liu, Y.; Avdalovic, N. Anal. Chem. 1999, 71, 2774-2781. (10) Liang, L.; Mo, S.; Cai, Y.; Mou, S.; Jiang, G.; Wen, M. J. Chromatogr., A 2006, 1118, 134-138. (11) Jandik, P.; Cheng, J.; Avdalovic, N. J. Biochem. Biophys. Methods 2004, 60, 191-203. (12) Genzel, Y.; Ko ¨nig, S.; Reichl, U. Anal. Biochem. 2004, 335, 119-125. (13) Martens, D. A.; Loeffelmann, K. L. J. Agric. Food Chem. 2003, 51, 65216529. (14) Thiele, C.; Ga¨nzle, M. G.; Vogel, R. F. Anal. Biochem. 2002, 310, 171178. (15) Draisci, R.; Giannetti, L.; Boria, P.; Lucentini, L.; Palleschi, L.; Cavalli, S. J. Chromatogr., A 1998, 798, 109-116. (16) Rose, P. I. In Encyclopedia of Polymer Science and Engineering, 2nd ed.; Mark, H. F., Bikales, N., Overberger, C. G., Menges, G., Kroschwitz, J. I., Eds.; John Wiley & Sons, Inc.: New York, 1987; Vol. 7, pp 488-513. (17) Gelatin Manufacturers Institute of America, Inc. Gelatin; New York, 1993. (18) Nicholas, N. C.; Welsch, J. R. Ballistics Gelatin. Institute for Non-lethal Defense Technologies Report; Applied Research Laboratory, The Pennsylvania State University, 2004. (19) Prockop, D. J. In Encyclopedia of Biological Chemistry; Lennarz, W. J., Lane, M. D., Eds; Academic Press: New York, 2004; pp 482-487. (20) Eastoe, J. E. Biochem. J. 1955, 61, 589-600. (21) Neuman, R. E.; Logan, M. A. J. Biol. Chem. 1950, 186, 549-556.

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tool in species identification of the collagenous raw material source. This may be especially true when comparing warm- and cold-blooded animals or animals from warm and cold waters. There are a number of derivatization reagents and methods specific for primary amine/amino acids. Reagents or methods specific for secondary amine/amino acids are less numerous. Early techniques used colorimetric methods for selective determination of 4-hydroxyproline by oxidation followed by reaction with pdimethylaminobenzaldehyde to form a highly colored adduct.23,24 The reaction, although very specific for hydroxyproline, is very sensitive to reaction conditions and reproducibility was often an issue.25 Precolumn derivatization with reagents that produce fluorescent adducts has been used for determination of secondary amino acids in urine and tissue samples.26-28 However, these reagents lack specificity for secondary amino acids and require a degree of kinetic control during derivatization to improve selectivity. Another technique commonly used in HPLC applications has been to block or “mask” primary amino acids by derivatization with o-phthalaldehyde (OPA) in the presence of a thiol followed by an additional derivatization with a reagent to produce fluorescent29-36 or electrochemically active adducts.37 Although these approaches have been successful, stability of the amino acid derivatives remains an issue. The use of IPAD eliminates the stability issues often encountered when using fluorescence detection in amino acid analyses. Detection is dependent upon oxidation of the amine functionality, not the stability of the fluorescent isoindole product. The need for a second derivatization during sample preparation adds to the overall analysis time. Previous work has shown that secondary amino acids can be selectively determined by removing OPA-derivatized primary amino acids prior to HPLC analysis through solid-phase extraction (SPE) with C18 packed columns.38-41 The remaining proline and 4-hydroxyproline were then derivatized with fluorescent reagents and detected by fluorescence after separation by reversed-phase HPLC. The use of SPE can reduce the time needed for sample (22) Privalov, P. L. In Advances in Protein Chemistry; Anfinsen, C. B., Edsall, J. T., Richards, F. M., Eds; Academic Press: New York, 1982; Vol. 35, pp 55-87. (23) Neuman, R. E.; Logan, M. A. J. Biol. Chem. 1950, 184, 299-306. (24) Stegemann, H. Hoppe-Seylers Z. Physiol. Chem. 1958, 311, 41-45. (25) Bergman, I.; Loxley, R. Anal. Chem. 1963, 35, 1961-1965. (26) Ahnoff, M.; Grundevik, I.; Arfwidsson, A.; Fonselius, J.; Persson, B.-A. Anal. Chem. 1981, 53, 485-489. (27) Hughes, H.; Hagen, L.; Sutton, R. A. L. Clin. Chem. 1986, 32, 1002-1004. (28) Kakinuma, M.; Watanabe, Y.; Hori, Y.; Oh-I, T.; Tsuboi, R. J. Chromatogr., B 2005, 824, 161-165. (29) Einarsson, S. J. Chromatogr. 1985, 348, 213-220. (30) Whiteside, I. R. C.; Worsfold, P. J. Anal. Chim. Acta 1988, 204, 343-348. (31) Nathans, G. R.; Gere, D. R. Anal. Biochem. 1992, 202, 262-267. (32) Ikeda, M.; Sorimachi, K.; Akimoto, K.; Okazaki, M.; Sunagawa, M.; Niwa, A. Amino Acids 1995, 8, 401-407. (33) Sormiachi, K.; Ikeda, M.; Akimoto, K.; Niwa, A. J. Chromatogr., B 1995, 664, 435-439. (34) Castelain, S.; Kamel, S.; Picard, C.; Desmet, G.; Sebert, J. L.; Brazier, M. Clin. Chim. Acta 1995, 235, 81-90. (35) Mazzi, G.; Fioravanzo, F.; Burti, E. J. Chromatogr., B 1996, 678, 165-172. (36) Hutson, P. R.; Crawford, M. E.; Sorkness, R. L. J. Chromatogr., B 2003, 791, 427-430. (37) Williams, C. K.; Koppang, M. D. Electroanalysis 2006, 18, 2121-2127. (38) Inoue, H.; Date, Y.; Kohashi, K.; Yoshitomi, H.; Tsuruta, Y. Biol. Pharm. Bull. 1996, 19, 163-166. (39) Inoue, H.; Kohashi, K.; Tsuruta, Y. Anal. Chim. Acta 1998, 365, 219-226. (40) Tsuruta, Y.; Inoue, H. Anal. Biochem. 1998, 265, 15-21. (41) Inoue, H.; Iguchi, H.; Kono, A.; Tsuruta, Y. J. Chromatogr., B 1999, 724, 221-230.

preparation when compared to liquid-liquid extractions aimed at removing primary amino acids from the sample matrix. This paper presents a selective method for secondary amino acid analysis in gelatin hydrolysates using a scheme to remove primary amino acids by utilizing an OPA/dithiol derivatization followed by passage through C18 packed SPE columns. Anionexchange HPLC was used to separate the remaining secondary amino acids under isocratic conditions, followed by IPAD. The selectivity and sensitivity of this assay allowed for the precise and accurate quantitation of secondary amino acids in gelatin hydrolysates. The high precision of this method enabled the distinction of gelatins extracted from different raw material sources based on the relative amounts and ratios of secondary amino acids present in their acid-digested hydrolysates EXPERIMENTAL SECTION Reagents. The amino acids trans-4-hydroxy-L-proline (>99%) and DL-proline (99%) were obtained from Aldrich. DL-Dithiothreitol (DTT, Cleland’s reagent, >99%), OPA (HPLC grade, dmt, 99%), sodium tetraborate decahydrate (Reagent Plus, 99.5-105.0%), and acetonitrile (ChromSolvePlus, HPLC, >99.9%) were obtained from Sigma-Aldrich. Sodium hydroxide (50% w/w) and hydrochloric acid (ACS grade) were obtained from Fisher Scientific. All aqueous solutions were prepared from 18.2 MΩ‚cm water purified by a NANOpure DIamond (Barnstead) deionization system. Water was degassed by using compressed helium obtained from LinWeld (Sioux City, IA). The OPA/DTT solution was prepared by adding 0.0200 g of DTT to a 200-mL volumetric flask and dissolving it in ∼150 mL of 10 mM borate buffer. OPA in the amount of 0.0160 mg was dissolved in 3 mL of acetonitrile and was quantitatively transferred to the volumetric using an additional 2 mL of acetonitrile. The contents were diluted to the mark with the borate buffer. A series of aqueous standards containing proline and hydroxyproline were made by adding 0.0500 g of proline and 4-hydroxyproline to a 500-mL volumetric flask and filling to the mark with deionized water (100 parts per million, ppm). Gelatin Hydrolysate Sample Preparation and Derivatization. Commercial and experimental gelatin samples were provided by GELITA USA, Inc. (Sioux City, IA). Gelatin types provided include the following: type B bovine bone, type B bovine hide, type A porcine skin, type A catfish skin, type A Nile perch skin, and type A tilapia skin. Gelatin hydrolysates were prepared by drying the gelatin samples in a drying oven at 105 °C for 24 h. Each hydrolysis corresponds to a single value of n, which also corresponds to the number of days tested. Samples weighing 1.000 g were placed into a 250-mL Erlenmeyer flask and 50 mL of 6 M HCl was added. The solution was hydrolyzed for 24 h at 110 °C. The hydrolyzed gelatin solution was quantitatively transferred to a 1-L volumetric flask with water to make a 0.1% gelatin solution. An additional dilution was performed to obtain a 0.01% gelatin solution. Some analyses required additional dilutions. To a test tube, 3 mL of the OPA/DTT solution was added followed by 1 mL of 0.01% hydrolyzed gelatin solution and 2 mL of water. The test tube was vortexed for 5 s. The derivatization reaction was immediate. However, the next step was not carried out for at least 5 min after the initial mixing. The pH of the derivatized gelatin solution was adjusted from approximately pH ) 9-9.1 to below pH ) 8.0 by adding ∼15 µL of 5 M HCl using an adjustable micropipet. A 3-mL PrepSep-C18 SPE column (500 mg of C18,

Table 1. Dionex Optimized Waveform for Detection of Amino Acids Using a Au Working Electrode in an Alkaline Mobile Phase (Potential vs Ag/AgCl) time/s

potential/V

0.00 0.04 0.05 0.21 0.22 0.46 0.47 0.56 0.57 0.58 0.59 0.60

-0.20 -0.20 0.00 0.00 0.22 0.22 0.00 0.00 -2.00 -2.00 0.60 -0.20

start integration

end integration

Fisher Scientific) was prepared by passing 2 mL of acetonitrile followed by 2 mL of water through the column. The solution containing the derivatized gelatin was slowly passed through the SPE column to remove the derivatized amino acids, excess OPA, and excess DTT. Fractions eluted from the SPE column were collected in 0.5-mL increments. However, after the ideal volume for elution of secondary amino acids from the SPE column had been established, the first 2.0 mL of eluent was discarded. Analyses were performed on fractions collected between 2.0 and 3.0 mL. The collected fractions were added to Dionex AD40 autosample vials and capped with Dionex filter caps. Chromatography and Instrumentation. A modular Dionex chromatography system equipped with Chromeleon 6.54 software management system was used for chromatographic control and analysis. The system was equipped with a Dionex amperometry cell with a 1-mm-diameter Au working electrode used with an ED50A trimode electrochemical detector. A Dionex combination pH/Ag/AgCl reference electrode was used for all voltammetric measurements. The electrochemical response acquired from IPAD is given in coulombs as a consequence of integration of current and is recorded in this manner by the Dionex Chromeleon software. Alternatively, the integrated response could be divided by the integration time to yield an average current.6 Dionex AminoPac PA10 2 × 50 mm guard and 2 × 250 mm analytical anion-exchange columns were installed along with the amperometry cell in a Dionex LC25 column oven maintained at 30 °C. The mobile phase consisting of 100 mM NaOH was degassed with helium and maintained under helium for the duration of the experiments. A flow rate of 0.25 mL/min was used and the injection volume was 25 µL, unless otherwise stated. A Dionex preprogrammed waveform optimized for amino acid detection at Au electrodes with their AminoPac PA10 column was used for the electrochemical detection. This waveform is given in Table 1. The rationale for the design of a waveform, similar to that given in Table 1, has been discussed in detail.8 RESULTS AND DISCUSSION Analysis of Hydroxyproline and Proline. In developing a selective assay for secondary amino acids, we decided to test our HPLC-IPAD detection using the waveform in Table 1 with aqueous standard solutions containing 4-hydroxyproline and proline. A chromatogram of a standard solution containing 2.00 ppm Analytical Chemistry, Vol. 79, No. 17, September 1, 2007

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Figure 2. Chromatogram of a 0.01% gelatin hydrolysate solution. Chromatographic conditions as stated in Figure 1, 10-µL injection: (1) 4-hydroxyproline; (2) proline. Inject at t ) 0 min with 0.5-min signal acquisition delay. Figure 1. Representative chromatogram of a 2.00 ppm standard solution containing (1) 4-hydroxyproline and (2) proline analyzed on a Dionex AminoPac PA10 column with a 100 mM NaOH mobile phase pumping at 0.25 mL/min with a 25-µL injection. Inject at t ) 0 min with 0.5-min signal acquisition delay. Table 2. Statistical Results of the Least-Squares Best-Fit Line of 4-Hydroxyproline and Proline Standards Comprising the Calibration Curvesa statistic

4-hydroxyproline

proline

no. of pts eq best fit corr coeff std err of est std err slope

8 y ) (21.590)x + 1.052 0.9992 1.016 nC‚min 0.347 nC‚min/ppm

8 y ) (14.543)x + 0.398 0.9997 0.443 nC‚min 0.151 nC‚min/ppm

a

The relationship represents a plot of peak area (nC‚min) vs concentration (ppm).

4-hydroxyproline and proline shows near-baseline separation (Figure 1) under isocratic conditions using a flow rate of 0.25 mL/ min and a 100 mM NaOH mobile phase using the Dionex AminoPac PA10 column. A series of standards ranging from 0.05 to 3.00 ppm of each secondary amino acid produced a linear detector response when peak areas are plotted versus concentration (Table 2). The detection limits are ∼10 parts per billion (ppb) for standard solutions of 4-hydroxyproline and proline, respectively. This corresponds to a detection limit of 1.9 × 10-12 and 2.2 × 10-12 mol (25-µL sample injection) of 4-hydroxyproline and proline solutions. After the successful analysis of amino acid standard solutions, we turned our attention to the analysis of gelatin hydrolysates. A chromatogram of a gelatin hydrolysate solution is presented in Figure 2. As expected, primary amino acids were detected along with secondary amino acids using IPAD. The peak corresponding to 4-hydroxyproline appeared asymmetrical and was preceded by a smaller shoulder peak that interfered with the integration of 4-hydroxyproline. A series of experiments were conducted in which the flow rate and mobile-phase concentration were adjusted in an attempt to improve peak symmetry and resolve the shoulder peak from 4-hydroxyproline. When the flow rate was slowed and the concentration of NaOH in the mobile phase reduced, a coeluting peak was seen emerging from the 4-hydroxyproline peak 6618 Analytical Chemistry, Vol. 79, No. 17, September 1, 2007

Figure 3. Chromatograms of 0.01% gelatin hydrolysate solutions with 10-µL injections under the following chromatographic conditions: (A) 100 mM NaOH, 0.25 mL/min, (B) 75 mM NaOH, 0.20 mL/ min, and (C) 50 mM NaOH, 0.15 mL/min. (1) 4-hydroxyproline, (2) proline, and (3) unknown interferent. Inject at t ) 0 min with 0.5-min signal acquisition delay.

(Figure 3). The interfering peak could not be successfully resolved without substantial increases in analysis time and the use of a mobile-phase gradient. We suspected that primary amino acids were the source of the interfering peaks. Instead of making substantial changes to the chromatographic conditions, we opted to develop a scheme to selectively remove primary amines prior to chromatographic analysis. We hoped this would keep the chromatography simple while minimizing overall time of analysis. The use of an OPA/ DTT derivatization followed by SPE proved to be very effective at removing primary amino acids. The interfering peaks were successfully removed, and peak shape and resolution of the remaining secondary amino acids were improved. The removal of primary amines through derivatization and SPE had a large impact on the quantitation of secondary amino acids in gelatin hydrolysates. Figure 4 shows a chromatogram of a gelatin hydrolysate with and without the OPA/DTT/SPE treatment. The interfering peak coeluting with 4-hydroxyproline inflated the 4-hydroxyproline content by 23.2%. The value of the proline peak increased nearly 3.7% after primary amine removal. The proline increase can be attributed to a more valid integration of peak area due to better peak-to-peak resolution and peak symmetry. Similar results were seen with all types of gelatin hydrolysates examined.

Figure 4. Chromatogram of a 0.01% bovine bone gelatin hydrolysate solution. Chromatographic conditions as stated in Figure 1, 25-µL injection. (A) No derivatization or SPE, (1) 4-Hpro ) 1.72 × 105 ppm; (2) Pro ) 1.30 × 105 ppm, (B) Primary amines removed by derivatization with OPA/DTT and SPE, (1) 4-Hpro ) 1.39 × 105 ppm; (2) Pro ) 1.35 × 105 ppm. Inject at t ) 0 min with 0.5-min signal acquisition delay.

Figure 6. Image of SPE column illuminated with UV light (366 nm) after passing ∼2 mL of OPA-derivatized gelatin hydrolysate solution (top). The fluorescent band contains derivatized primary amino acids. Within 1 h after use, the fluorescence intensity on the SPE column began to fade and a colored band became visible (bottom).

Figure 5. Peak area vs milliliters of derivatized bovine bone gelatin hydrolysate solution extracted through a C18 SPE column. Chromatographic conditions as stated in Figure 1, 25-µL injection. [ ) 4-hydroxyproline; 9 ) proline. Peak area for both 4-hydroxyproline and proline are maximized after ∼2.0 mL of solution has been extracted.

To reiterate our analysis scheme, we derivatized the gelatin hydrolysate with OPA in the presence of DTT, passed the entire mixture through a SPE column to remove the derivatized primary amino acids and analyzed the eluent with IPAD. Initially, we needed to determine eluent volumes through the SPE column in order to optimize secondary amino acid recovery and minimize OPA-derivatized primary amine and excess DTT elution from the SPE column. Optimal elution of the secondary amino acids from the SPE column occurred after ∼2 mL of solution had been passed (Figure 5). For most analyses, the first 2 mL of solution was discarded and the solution eluting between 2 and 3 mL was analyzed. The top image in Figure 6 shows a SPE column illuminated with a 366-nm UV lamp after an OPA-derivatized gelatin hydrolysate solution had been passed through the column. A strong, narrow fluorescent band near the top of the column indicated the presence of derivatized amino acid adducts. Within 1 h, the intensity of the fluorescent band diminished and became colored in visible light (bottom Figure 6). The amounts of proline and 4-hydroxyproline of gelatin hydrolysates of differing animal origin are presented in Figure 7. The data were generated by comparing results of the analysis of gelatin hydrolysates to the calibration curve created from the analysis of the amino acid standards. When significant sample preparation is a prerequisite for analysis, it is customary practice

Figure 7. Proline and 4-hydroxyproline content of gelatin hydrolysates extracted from various animal sources. n, the number of days tested. Error bars represent the 95% confidence interval calculated using the Student’s t. Light gray, 4-hydroxyproline; dark gray, proline. Bovine bone and hide gelatins are type B; other gelatins are type A.

to use internal standards to account for potential sample loss during the preparation of samples for analysis. We investigated three potential internal standards including trans-3-hydroxy-Lproline (Fluka), N-acetyl-L-proline (Fluka), and L-proline methyl ester hydrochloride (Aldrich). The amide- and ester-containing compounds suffered from degradation during the acid hydrolysis step. In addition to hydrolysis, the methyl ester eluted near the void volume due to the lack of an anionic functional group. The amino acid 3-hydroxyproline eluted near 4-hydroxyproline, and the two isomers were difficult to adequately resolve without substantial changes to mobile-phase flow rate and composition. Several other internal standards were considered, but their high cost and potential for hydrolysis during acid digestion dissuaded us from exploring their use. In lieu of a suitable internal standard, a series of experiments were conducted to examine possible sample loss during the sample preparation steps. Proline and 4-hydroxyproline were spiked into gelatin samples before and after hydrolysis. Gelatin samples spiked with proline, 4-hydroxyproline, or both before acid hydrolysis gave recoveries of 89-95 and 9299%, respectively. Samples spiked after hydrolysis gave recoveries Analytical Chemistry, Vol. 79, No. 17, September 1, 2007

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Figure 8. Ratio of 4-hydroxyproline to proline of gelatin hydrolysates extracted from various animal sources. n, the number of days tested. Error bars represent the 95% confidence interval calculated using the Student’s t. Bovine bone and hide gelatins are type B; other gelatins are type A.

of 94-97 and 96-103%, respectively. Most of the sample loss occurred during the hydrolysis of the gelatin samples. It is often valuable to know, or confirm, the raw material source from which gelatin has been extracted. Our analyses demonstrates that the ratio of 4-hyroxyproline to proline can distinguish the source of raw material when used in conjunction with the amounts of each secondary amino acid. The amounts of secondary amino acids found in collagen have been shown to differ among warmblooded animals based on body temperature and based on environmental temperature for cold-blooded animals.22 These naturally occurring differences in secondary amino acid content may be used to aid in identification of the gelatin raw material source. Figure 8 shows the mean ratios of 4-hydroxyproline to proline in the gelatin hydrolysate samples. For each gelatin sample, the ratio of 4-hydroxyproline to proline content was measured from the IPAD chromatogram. The bovine bone, bovine hide, porcine skin, and Nile perch skin gelatin hydrolysate samples all contained mean amounts of proline that were statistically indistinguishable with a 95% confidence level (Figure 7). However, the four gelatins could be distinguished with a 99.9% confidence level by comparing the mean amounts of 4-hydroxyproline and by comparing the mean ratio of secondary amino acids. Similarly, catfish skin and tilapia skin gelatin hydrolysates have indistinguishable mean amounts of 4-hydroxyproline and proline with a 95% confidence level. The catfish skin and tilapia skin gelatin hydrolysates can be distinguished at the 95% and the 99% confidence level by comparing the mean ratio of secondary amino acids. In most cases, gelatin types could be discriminated with a confidence level of g99% by comparing the ratio of secondary amino acids. By knowing the mean amount of 4-hydroxyproline, proline, and the ratio of the two secondary amino acids, every gelatin type tested could be distinguished from one another with a confidence level g99%. A more detailed statistical analysis of information provided in Figures 7 and 8 along with calculated t-values and confidence levels can be found in the Supporting Information. There are, however, caveats to consider for this type of analysis. First, the purity of the gelatin has to be reasonably known. Processing conditions can greatly influence the amounts 6620 Analytical Chemistry, Vol. 79, No. 17, September 1, 2007

of noncollagenous protein, ash, and moisture in the samples leading to inconsistent values of the absolute amounts of secondary amino acids. Discrepancies in the analysis of secondary amino acids due to gelatin purity may be mitigated by examining the ratio of secondary amino acids. While the absolute amounts of secondary amino acids may change with sample purity, the ratio gives a more robust indicator of raw material origin due to the relativistic nature of the measurement. A second caveat is the assumption that the gelatin under analysis is from a single species, not a mixture of two or more. SPE Efficiency and Derivatization Reagent Selection. A very broad, often split peak with a retention time of 10 min was observed in the LC-EC process in addition to the proline and hydroxyproline signals and is tentatively assigned to excess DTT and derivatized amino acids that were not removed by solid-phase extraction. However, elution of proline and 4-hydroxyproline from the SPE column could be controlled before elution of the unwanted products begin to bleed from the SPE column when appropriate elution conditions were selected. Improved retention of both of the unwanted products on the SPE column was achieved by lowering the pH before the solid-phase extraction step. At the derivatization pH (pH ) 9.1), DTT and the derivatized products were less effectively retained on the SPE column tending to elute early into the collected fractions (after passing ∼1-1.5 mL of derivatized solution). By lowering the pH < 8, the unwanted substances were effectively retained on the SPE column. At a pH near 9, potential dissolution of the silica-based end-capped ODSC18 material is not considered to be a determining factor on the retention given the small column volumes of material passed through SPE.42 Instead, the pH dependency arises from dithiothreitol pKa’s of 9.2 and 10.1.43 At a pH of 9.1, appreciable amounts of the dithiothreitol will be deprotonated and as such, hydrophilic and less likely to bind to the SPE column. Likewise, the free thiol on the OPA derivatives will be deprotonated and more hydrophilic. Removal of primary amines by SPE may be enhanced by changing derivatization reagents from OPA to naphthalene-2,3-dicarboxaldehyde (NDA). The additional ring system of NDA and its amino adducts should result in increased hydrophobicity as compared to the OPA adducts. NDA has a pH-rate profile similar to that of OPA.44 The time required for derivatization should be similar. Although other nucleophiles may be better suited for removal by SPE, DTT was chosen as the nucleophile due to its low odor. DTT has also been successfully used for primary amine analysis with both fluorescence and UV detection.45-47 Although, OPA/ DTT amino acid adducts exhibit decreased fluorescence compared to more traditionally used thiols such as β-mercaptoethanol,48 it is inconsequential since this method removes the OPA derivatives by SPE before IPAD. The use of a dithiol presents the possibility of forming dimer adducts, increasing the hydrophobicity of (42) Kirkland, J. J.; van Straten, M. A.; Claessens, H. A. J. Chromatogr., A 1995, 691, 3-19. (43) Whitesides, G. M.; Lilburn, J. E.; Szajewski, R. P. J. Org. Chem. 1977, 42, 332-338. (44) de Montigny, P.; Stobaugh, J. F.; Givens, R. S.; Carlson, R. G.; Srinivasachar, K.; Sternson, L. A.; Higuchi, T. Anal. Chem. 1987, 59, 1096-1101. (45) Vurek, G. G. Anal. Chem. 1982, 54, 840-842. (46) Nielsen, P. M.; Petersen, D.; Dambmann, C. J. Food Sci. 2001, 66, 642646. (47) Simons, S. S., Jr.; Johnson, D. F. J. Org. Chem. 1978, 43, 2886-2891. (48) Chen, R. F.; Scott, C.; Trepman, E. Biochim. Biophys. Acta 1979, 576, 440445.

derivatized amino acids, and ultimately increasing primary amine extraction efficiency by SPE. However, reaction conditions in these experiments do not favor dimer formation.47 The hydrolysis conditions may play a factor in the recovery of proline and 4-hydroxyproline from the hydrolysate. Recent research has shown that a 1-h alkaline hydrolysis in an autoclave at 121 °C leads to a much higher recovery of secondary amino acids compared to a 16-h acidic hydrolysis at 110 °C in gelatin hydrolysates.49 This may be an important factor to consider when comparing secondary amino acid data among different methods. CONCLUSIONS The removal of primary amino acids prior to analysis greatly improved method precision and accuracy and decreased the analysis time of secondary amino acids using anion-exchange HPLC-IPAD. Without the removal of primary amino acids, a mobile phase gradient or a flow rate decrease would be required to get sufficient resolution between the interfering peak and 4-hydroxyproline using the AminoPac column. The removal of primary amino acids allows the use of an isocratic mobile phase with a flow rate that keeps the chromatographic analysis time less than 8 min, including time needed for baseline equilibration. The use of IPAD eliminated the need for a second derivatization for detection, decreasing the time and complexity required for sample preparation. The overall result is the selective, sensitive, and quick assay of the remaining secondary amino acids. Limits of detection for the secondary amino acids approach 10 ppb (∼2 pmol/ injection). The selectivity afforded by this technique greatly (49) Badadani, M.; SureshBabu, S. V.; Shetty, K. T. J. Chromatogr., B 2007, 847, 267-274.

improves the ability to accurately quantify the amount of secondary amino acids in gelatin hydrolysates. Comparison of the amounts and ratios of secondary amino acids can distinguish gelatin hydrolysates extracted from bovine, porcine, and several fish sources. The ratio of 4-hydroxyproline to proline proved to be a more reliable means to distinguish gelatin raw material sources and provided a greater degree of statistical certainty in the comparisons. An additional benefit for this technique is the potential for use in high-throughput applications. Several sample preparation steps are amenable to automation (SPE vacuum manifolds, fraction collectors) and the use of versatile, modular chromatography instruments aid in making the analysis quick, cost-effective and ideally suited for quality assurance-type applications. ACKNOWLEDGMENT The authors acknowledge the University of South Dakota Department of Chemistry, including Benjamin Lamprecht and Sheereene Hussain. We also thank Celia Williams for preliminary discussions regarding OPA derivatization. A very special thanks is given to GELITA USA, Inc., for providing financial support, facilities, and expertise throughout this research. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review April 23, 2007. Accepted June 24, 2007. AC070819W

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