Quantitative Analysis of TAG in Oils Using Lithium Cationization and Direct Infusion ESI Tandem Mass Spectrometry

Transcription

1 J Am Oil Chem Soc (2015) 92: DOI /s ORIGINAL PAPER Quantitative Analysis of TAG in Oils Using Lithium Cationization and Direct Infusion ESI Tandem Mass Spectrometry Louis Ramaley Lisandra Cubero Herrera Jeremy E. Melanson Received: 19 September 2014 / Revised: 7 January 2015 / Accepted: 21 January 2015 / Published online: 6 February 2015 AOCS 2015 Abstract This study was undertaken to determine whether triple-stage mass spectrometry (MS 3 ) could be employed to obtain quantitative and regioisomeric data from complex oil samples without the need for a chromatographic step in the analysis protocol. Lithium-7 trifluoroacetate and electrospray ionization were used to form lithium adducts of the triacylglycerols (TAG) in a fish oil sample. The first-generation precursor ion was the lithium-tag adduct, the second-generation precursor ion was formed by loss of a neutral acid side chain in the first fragmentation. The ions used for analysis were formed in the second fragmentation by loss of the lactones of the acid side chains remaining after the first fragmentation. This analysis scheme provided quantitative and regioisomeric data without interference from TAG in the sample other than TAG with the same acyl carbon number, one more double bond, Electronic supplementary material The online version of this article (doi: /s ) contains supplementary material, which is available to authorized users. L. Ramaley (*) Department of Chemistry, Dalhousie University, Halifax, NS B3H 4R2, Canada louis.ramaley@dal.ca L. Ramaley L. C. Herrera J. E. Melanson National Research Council Canada, 1411 Oxford St., Halifax, NS B3H 3Z1, Canada Present Address: L. C. Herrera Canadian Food Inspection Agency, 1992 Agency Dr., Dartmouth, NS B3B 1Y9, Canada J. E. Melanson Measurement Science and Standards, National Research Council Canada, 1200 Montreal Rd., Ottawa, ON K1A 0R6, Canada and two acyl side chains in common with the analyte. Even in this case a majority of the interferences could be estimated and compensated. Analysis of synthetic samples containing the fish oil matrix indicated that both absolute and relative quantitative data could be obtained with average errors of approximately 5 %. The method proved well suited to routine analyses of complex oil samples. Keywords Triacylglycerols Quantitative oil analysis Tandem mass spectrometry Direct infusion Lithium adducts Introduction Most plant and animal oils, e.g., olive or salmon oils, consist mainly of mixtures of triacylglycerols (TAG). The physical and biochemical properties of TAG depend not only on the types of fatty acids in the molecule, but also the position of substitution along the glycerol backbone, the two outer positions being designated as sn-1 and sn-3 and the inner position as sn-2. The existence of acyl side chains at different positions and the large number of different fatty acids available for esterification lead to the possibility of a wide variety of natural TAG. Thus the development of analytical methods that can determine both the fatty acid content of the various TAG in an oil and the position of substitution (regioisomerism) is very important. At present, exhaustive analysis of the components in oils is usually accomplished using some form of chromatography as a separation technique coupled to mass spectrometry to provide additional separation and specific identification of TAG components [1 5]. Including chromatography in the analysis protocol provides a separation step which can reduce analyte overlap, noise, and ion suppression

2 324 J Am Oil Chem Soc (2015) 92: effects. Chromatography requires extra time, affecting throughput, and additional experimental steps, which may decrease reproducibility. It also limits the time of analysis to the chromatographic peak width, lowers the analyte concentration, and may cause matrix and ionization sensitivity changes due to gradient changes in the chromatographic eluent. Mass spectrometry (MS) alone also provides analyte separation by molecular mass and fragmentation and has been used directly to investigate lipid composition, especially of glycerophospholipids, in biological samples [6 10] and plays a major role in shotgun lipidomics [11]. Specific analysis of TAG in biological samples has also been accomplished using MS alone [12 23]. The majority of these applications are qualitative in nature, but some have produced quantitative data [12, 15, 16, 21] and some have been applied specifically to oils [18, 19, 21]. Several different ionization techniques have been employed when applying MS to the direct analysis (without chromatography) of TAG. The most popular of these are atmospheric pressure chemical ionization (APCI), electrospray ionization (ESI), matrix assisted laser desorption ionization (MALDI), and negative ion chemical ionization (NICI). APCI produces both [A+H] + protonated molecules and [A+H RCOOH] + fragment ions with the loss of one of the acyl side chains, the former providing molecular mass data and the latter data on side chain composition. The abundance of [A+H] + ions depends on the saturation level of the TAG and is very low for saturated TAG. The relative abundances of the [A+H RCOOH] + ions are dependent on the positions of the fatty acids on the glycerol backbone, with the sn-2 fatty acid being lost less favorably than the sn-1 or sn-3 fatty acids, providing information on regioisomerism. Complex mixtures are difficult to analyze using this approach, and attempts to improve selectivity by performing collision-induced dissociation (CID) of [A+H] + ions have not been successful [1]. TAG with acyl chains containing four double bonds or more failed to provide abundance differences [24]. For these reasons, APCI, when operating in the single-stage MS mode, may be suited for the investigation of simple TAG mixtures, but not for direct quantitative analysis of complex oil samples. MALDI with time-of-flight (TOF) mass spectrometers [19, 22, 23] produces protonated molecules or cationized adducts. To obtain structural information requires fragmentation and a second stage of mass analysis, usually provided by a second TOF mass analyzer. Given the rather poor precision of the MALDI technique for quantitative analysis and the rarity of TOF/TOF instruments, this technique is not well suited for routine, high-throughput, quantitative studies of TAG mixtures. The NICI technique applied directly to TAG mixtures uses NH 3 to form [A H] ions [1, 12, 16, 18]. Using dual-stage or tandem MS (MS 2 ), [RCOO] and [A H RCOOH 100] fragment ions are formed, the abundances of the former are not position sensitive and are used for quantification while the abundances of the latter depend on the position of substitution on the glycerol backbone and can be used to determine regioisomerism. Samples are volatilized from a rhenium-wire direct-insertion probe. Sampling time is limited and sampling involves several steps where imprecision can arise, which can limit this technique for high-throughput, direct, quantitative analysis of TAG mixtures. Conventional ESI and its direct atmospheric sampling variants such as desorption electrospray ionization (DESI) [25] and probe electrospray ionization (PESI) [26] produce adducts with alkali or ammonium ions. If no ions are added to the samples, [A+Na] + with perhaps some [A+K] + adducts are formed from ions intrinsic to the sample or solvent. Ammoniated adducts [13, 17, 21] and lithiated adducts [14, 15, 20] are formed by adding appropriate salts to the sample solutions. In the absence of fragmentation single-stage MS can separate TAG only by the total acyl carbon number (ACN) and the double bond number (DBN), providing no information on side chain identity or position. ESI, although a soft ionization technique, usually provides some fragmentation of TAG, and this can be enhanced by source voltage settings (in-source fragmentation). Thus single-stage ESI MS might provide information on side chain identity and substitution position. Unfortunately the same fragment ions can be produced by two or more different TAG, a situation very likely in a complex oil sample, confusing the analysis. This can be avoided through the use of double-stage MS. When using ESI, CID results in the loss of one of the acyl side chains, producing [A+NH 4 NH 3 RCOOH] + ions for ammoniated TAG and both [A+Li RCOOH] + and [A+Li RCOOLi] + ions for lithiated TAG. As observed with other ionization methods, the side chain loss is more favored from the sn-1/3 positions than from the sn-2 position. Thus ESI MS 2 will separate TAG according to their ACN and DBN, identify the acyl side chains by molecular mass, and provide regioisomeric information for simple mixtures. However, when two isomers with one side chain in common are present, peak overlap will occur which may prevent accurate analysis. For example, consider the case where TAG 16:0/16:0/18:0 and 16:0/14:0/20:0 are both present with 16:0 representing palmitic acid, etc. For both lithium adducts the precursor ion m/z is (for the ions containing the heavier lithium-7 isotope). The MS 2 spectrum of 16:0/16:0/18:0 will have product ions at m/z and (loss of 16:0 and its lithium salt) and at m/z and (loss of 18:0 and its lithium salt), and the MS 2 spectrum of 16:0/14:0/20:0 will have product ions at m/z and (loss of 16:0 and its lithium salt), m/z and (loss of 14:0 and its lithium salt), and

3 J Am Oil Chem Soc (2015) 92: m/z and (loss of 20:0 and its lithium salt). The product ion peaks at m/z and are common to both TAG and the individual contributions cannot be determined, which may cause problems in quantification and assignment of regioisomerism. A similar analysis applied to ammoniated TAG again shows overlap of peaks. Since the overlapping peaks, both in the MS and MS 2 spectra, are due to isomers, high resolution instrumentation will be of no use in providing a solution. Thus, for complex mixtures such as fish oils, direct analysis using ESI MS 2 will not provide reliable analytical results. A second stage of CID (MS 3 ) might provide a solution to this problem. Hsu and Turk have studied the MS 3 spectra of lithiated TAG and noted that the spectrum of the second-generation precursor ion containing lithium, [A+Li RCOOH] +, exhibits fragment ions characteristic of the acyl side chains remaining after the first stage of CID, including lithiated acids, [RCOOH+Li] +, ions formed by the loss of a molecule which they interpreted to be an α,β-unsaturated acid (R =CHCOOH), [A+Li R 1 COOH R 2 =CHCOOH] +, and ions formed by the loss of a ketene, [A+Li R 1 COOH R 2 CH=C=O] + [14, 20]. The intensities of the lithiated acid ions are not strongly influenced by regioisomerism, but those of the other ions are. The intensities of the ions arising from the loss of an R =CHCOOH neutral fragment are quite position-dependent with loss of the acid from the sn-2 position being highly favored. These ions are also among the most intense ions in the spectra. In addition to the neutral losses being referred to as α,βunsaturated acids [14, 20], recent evidence suggests that the losses could actually be lactones [27]. For this reason and for simplicity this fragmentation will be referred to below as a lactone loss. Using the example of the 16:0/16:0/18:0 and 16:0/14:0/20:0 TAG above and taking as second-generation precursor the ion at m/z (loss of 16:0) where both TAG contributed, the MS 3 spectra of 16:0/16:0/18:0 will give peaks at m/z (loss of C16-lactone) and m/z (loss of C18-lactone) while that of 16:0/14:0/20:0 will give peaks at m/z (loss of C14-lactone) and m/z (loss of C20-lactone). Again, all m/z values assume the presence of the heavier lithium-7 isotope. Since there is no longer any overlap, the contribution of both TAG can be estimated, and positional information is available. A more detailed description of the MS 3 reaction sequence is presented in the supplementary material. The MS 3 spectra of ammoniated TAG have been reported by Byrdwell and Neff [28] to exhibit ions such as [RCO] +, [RCO H 2 O] +, [RCO+74] +, and [RCO+74 H 2 O] +, which are also indicative of the acyl side chains remaining after the first stage of CID. Hsu and Turk [20] observed the same ions and suggested that their abundances were positiondependent and could be used to study regioisomerism. 325 They also recommended the use of lithium as cationization reagent since the MS 3 spectra provided more information through charge remote fragmentation about the position of any double bonds in the acyl side chains. The MS 3 spectra of lithiated TAG has been used to obtain regioisomeric data in other studies [29 31]. Instruments capable of MS 3 are now relatively commonplace. Thus the study reported here was undertaken to determine whether the triple-stage mass spectrometry of lithiated TAG could be used to provide quantitative, regioisomeric data on TAG in complex oil matrices directly and quickly without the complexity of a chromatographic separation step. Materials and Methods Nomenclature and Abbreviations TAG regioisomers with different acyl side-chains are listed as AAB or ABC, where A, B and C represent different fatty acids. For example, AAB has fatty acid A at positions sn-1 and sn-2, and fatty acid B at position sn-3. Mass spectrometry cannot distinguish between the outer sn-1/3 positions and, thus, no distinction will be made between these positions. No attempt will be made to indicate double bond position or geometry. One-letter abbreviations will be used for fatty acids including: P, palmitic acid (16:0); S, stearic acid (18:0); and O, oleic acid (18:1). Lithium salts highly enriched in the heavier lithium-7 isotope were used in this study for reasons explained below. These produced results that were essentially monoisotopic in that no peaks were observed experimentally that could be assigned to ions containing the lighter lithium-6 isotope. When referring to ions formed from solutions of enriched lithium, the symbol 7 Li will be used, e.g., [A+2+ 7 Li] +, and in the rare cases were natural lithium is involved the symbol 6 Li will be employed. Thus the numeric value included within the square brackets indicates the increase in mass due to heavy isotopes in A. If this value is absent, it is assumed to be zero. Reagents and TAG Standards HPLC grade methanol, 2-propanol, and dichloromethane were purchased from Caledon Laboratories Ltd. (Georgetown, ON, Canada). Praxair (Halifax, NS, Canada) provided nitrogen gas (UHP). Lithium-7 carbonate (99.9 % 7 Li) was obtained from Cambridge Isotope Laboratories, Inc, (Andover, MA, USA) and trifluoroacetic acid (ReagentPlus) from Sigma-Aldrich Canada (Oakville, ON, Canada). The TAG standards OOS, PPO, POP, POS, PSO, and SPO were purchased from Larodan Fine Chemicals AB

4 326 J Am Oil Chem Soc (2015) 92: (Malmö, Sweden). All reagents were used as received without further purification. The fish oil matrix was provided by a Life Brands commercial Salmon and Fish Oil Supplement obtained at a local pharmacy. Primary stock solutions of TAG standards (~ mol L 1 ) were prepared in a dichloromethane:2- propanol:methanol solvent mixture (2:1:7, v/v/v). A primary stock solution of the fish oil matrix at the 1 mg ml 1 concentration level was prepared in the same solvent mixture. Secondary stock solutions were prepared by diluting the primary stock solutions with methanol. TAG standards were brought to concentrations of M and the fish oil to M, assuming an average molecular mass of 900 g mol 1. A stock solution of lithium-7 trifluoroacetate (LiTFA), the cationization reagent, ~ M, was prepared in methanol from stoichiometric amounts of lithium-7 carbonate and trifluoroacetic acid. Calibration and Sample Solutions All working solutions were prepared from the secondary TAG standard solutions and the LiTFA stock solution by dilution with methanol. All solutions contained M OOS as internal standard, M LiTFA, and M fish oil. The calibration solutions contained one of the analytes, PPO, POP, POS, PSO, or SPO, at one of the added concentrations, , , , , or M. Nine synthetic sample solutions were prepared by spiking the fish oil matrix with different combinations of concentrations of both PPO and POP. These were added at either the , , or M level. Twelve sample solutions were prepared with combinations of the three analytes, POS, PSO, and SPO, at the same added concentration levels. Mass Spectrometry All mass spectrometric measurements were made with an AB SCIEX 2000 QTRAP instrument (AB SCIEX, Foster City, CA, USA), a QqLIT mass spectrometer, equipped with a Turbo IonSpray source, operating in the ESI(+)MS 3 mode and running under Analyst V1.4 software. Regular instrument operating parameters were: mass range = m/z , resolution = unit mass, scan rate = 250 m/z per second, curtain gas = 30 psi, nebulizer gas = 20 psi, heater gas = 55 psi, IonSpray voltage = 4.8 kv, probe temperature = 100 C, declustering potential = 140 V, collision gas setting = high, collision energy = 45 ev, excitation energy = 50 V, excitation time = 150 ms, and linear ion trap (LIT) fill time = 20 ms. The instrument was operated using Q0 trapping to increase sensitivity. All solutions were introduced by direct infusion at 10 μl min 1 using a Chemyx, Inc (Stafford, TX, USA) Model Fusion 400 syringe pump. Each mass spectrum was acquired by integrating the instrument response over a period of 2.0 min and all measurements were made in triplicate. Both peak heights and peak areas were investigated as sources of quantitative data. Spectra, converted to ASCII text format by the data system, were examined by software written in-house. Peak heights were determined by leastsquares fitting to a Gaussian curve model and peak areas were determined by summing all the ion intensity in a window m/z 0.85 wide, centered on the peak maximum. The linear ion trap (LIT) resolution was well under unit mass. This software was used because it was available from prior projects, but is not necessary for the work described here, since instrument manufacturers provide software for peak identification and peak height and area determination. Other calculations are easily performed in a regular spreadsheet program. In general, data precision, slope standard deviation, and calibration curve linearity were all better with peak area data. Therefore, all data reported in this study were determined using peak areas. Results and Discussion Selection of Experimental Conditions TAG of the AAA type, where all acyl side chains are the same, do not exhibit regioisomerism and present little difficulty in analysis since the MS 3 spectrum is simple, only one calibration curve is required, and the ion selected for calibration can be any ion in the MS 3 spectrum, usually the most intense. For these reasons such a TAG was not included in this study. TAG of the AAB type with two different side chains do exhibit regioisomerism and present two unknowns, AAB and ABA, as measured by MS, for regioisomeric and quantitative analysis. Such analysis thus requires two equations with two unknowns and four different calibration curves. The ions selected for calibration should show intensity differences, depending on the position of substitution. TAG of the ABC type with no side chains in common are the most complex, exhibiting regioisomerism and presenting three unknowns, ABC, ACB, and BAC. Analysis in this case requires three equations with three unknowns and nine different calibration curves with the selected ions showing positional intensity differences. TAG of the AAB type are represented by PPO and POP in this study and TAG of the ABC type by POS, PSO, and SPO. OOS was used as the internal standard. These particular analytes and internal standard were selected because the concentrations of these TAG in the fish oil matrix are low, but not zero. This allows a wide range of concentrations to be examined for the analytes in

5 J Am Oil Chem Soc (2015) 92: the synthetic samples and causes few problems in keeping the concentration of internal standard the same in all the samples. The fish oil represents a complex natural oil which might pose analytical difficulties. A detailed description of the reaction schemes involved with the MS 3 of lithiated TAG and the manner in which ions are selected for analysis are presented in the supplementary material using POS as an example. Ideally the internal standard peaks should be recorded concurrently with the analyte peaks. In an MS 3 experiment this requires that the first- and second-generation precursor ions of the internal standard have the same m/z as those of the analyte, but that the final product ions are different. This requirement is quite difficult to meet for a single analyte and impossible if one internal standard is to be used for multiple analytes. The internal standard selected for this study did not meet this requirement and thus its data acquisition periods were set to bracket those of the samples or calibrants. With this arrangement there is little need to provide an expensive, isotopically enriched copy of the analyte as internal standard. A natural TAG that is reasonably similar to the analytes and is not present to any extent in the matrix will work as well. Comparisons of the data obtained in this study with and without the use of the internal standard demonstrated that use of the internal standard usually improved data precision, always improved the linearity of the calibration curves, and always improved the accuracy of determination of the analyte concentrations in the synthetic samples. Thus, use of an internal standard is mandatory in the method presented in this work. All data presented here were obtained using the m/z peak, [O+ 7 Li] +, in the MS 3 spectrum of OOS (m/z second-generation precursor ion) as internal standard. Lithium-7 was used as cationization reagent since natural lithium consists of about 7 % 6 Li and 93 % 7 Li and using natural lithium complicates both the spectra of lithiated species and correcting for peak overlap by higher peaks in the isotope cluster of interferents as discussed below. The concentration of cationization reagent was held at M which was high enough to provide high concentrations of lithiated TAG. Some sodiated TAG were also observed. This concentration avoided instrument contamination and formation of large amounts of clusters of lithium with the trifluoroacetate anion. Lithium-7 compounds are readily available and relatively inexpensive to use at this concentration level. Calibration Preliminary trials indicated that, even with an internal standard, the effect of the fish oil matrix was so strong that the slopes of the calibration curves were completely 327 different in the presence and absence of the matrix. Thus all calibration was performed with solutions containing the matrix, which could result in ion suppression. Ion suppression can occur in ESI due to signal saturation at high concentration levels and it has been suggested that analyte concentrations be held at around the M level to avoid this [32, 33]. In the case of TAG, ion suppression can also be caused by aggregation of the TAG which can be avoided at TAG concentrations around the M level or below [8, 11]. Nilsson and Skansen [34] have shown that, for ionization limited systems as opposed to detector limited systems, the use of an internal standard should provide linear calibration curves. For these reasons, the concentrations of the TAG in our solutions were held around the M level. Calibration in the presence of the matrix will almost always involve a background concentration of the analyte. This will not affect the slope of the calibration curve but will lead to a non-zero intercept. This does not present a problem as long as it can be assumed that the instrument response will be zero in the absence of analyte, since only the slopes are used to obtain the quantitative data. This assumption is not particularly good for single-stage MS where there is significant background noise, but is reasonable for MS 3 where most, if not all, of the background has been filtered out. As mentioned above the first-generation precursor ion is the lithiated TAG, [A+ 7 Li] +, (m/z for PPO/POP and m/z for POS/PSO/SOP). The second-generation precursor ion, [A+ 7 Li RCOOH] +, arises from the loss of a neutral acid side chain. There are two choices of secondgeneration precursor ion for PPO/POP, loss of P, m/z 583.5, and loss of O, m/z 557.5, and three choices for POS/PSO/ SPO, loss of P, m/z 611.6, loss of O, m/z 585.5, and loss of S, m/z The major ions in the MS 3 spectra are lithiated acids, lithiated ions formed by the loss of the lactones of the remaining acyl side chains and lithiated ions formed by the loss of the ketenes of the remaining acyl side chains. An example of the MS 3 spectra for POS/PSO/SPO is shown in Fig. 1. In Fig. 1 the ions at m/z 289 and 291 correspond to the lithiated acids [O+ 7 Li] + and [S+ 7 Li] +, which show good intensity but with little sensitivity to substitution position. The ions at m/z 329 and 331 are formed by the loss of an S-lactone and O-lactone, respectively. These ions are strongly position-dependent, being much more abundant when the lactone lost corresponds to the acid originally in the sn-2 position (Fig. 1a, b). The intensities are intermediate when the original loss of the neutral acid was from the sn-2 position (Fig. 1c). The ions at m/z 345 and 347 are formed by the loss of an S-ketene and O-ketene, respectively. The intensities of these ions are also positiondependent, being higher when the ketene lost corresponds to the acid originally in the sn-1/3 positions. The intensities

6 328 J Am Oil Chem Soc (2015) 92: Fig. 1 MS 3 spectra of a POS, b PSO, and c SPO. In each case the first-generation precursor ion is m/z and the secondgeneration precursor ion, m/z 611.6, has lost a neutral P acid side chain a b c Table 1 Calibration parameters for PPO and POP using fragmentation losses of both lactones and ketenes TAG 2nd Precursor Ion observed Slope SD r 2 m/z Loss m/z Loss PPO P P-lactone O-lactone O P-lactone POP P P-lactone O-lactone O P-lactone PPO P P-ketene O-ketene O P-ketene POP P P-ketene O-ketene O P-ketene are intermediate when the original neutral acid loss was from the sn-2 position (Fig. 1c). The best choice of masses for quantification would be those of the ions formed by lactone loss, both from an intensity and position-selective perspective. The ions formed by loss of a ketene would be expected to provide less satisfactory results, but were also investigated in this study. Tables S1 and S2 in the supplementary material list all the ions and ratios of mass to charge involved in the calibration. As shown in these tables there are six separate calibrations possible for PPO/POP and eighteen calibrations for POS/PSO/SPO. Table 1 lists slopes, standard deviations (SD), and squares of the correlation coefficient (r 2 ) pertaining to the calibration curves for PPO and POP for both the lactoneand ketene-loss ions. Table 2 lists these parameters for POS, PSO, and SPO for the lactone-loss ions and Table 3

7 J Am Oil Chem Soc (2015) 92: Table 2 Calibration parameters for POS, PSO, and SPO using fragmentation losses of lactones TAG 2nd Precursor Ion observed Slope SD r 2 m/z Loss m/z Loss POS P O-lactone S-lactone O P-lactone S-lactone S P-lactone O-lactone PSO P O-lactone S-lactone O P-lactone S-lactone S P-lactone O-lactone SPO P O-lactone S-lactone O P-lactone S-lactone S P-lactone O-lactone Table 3 Calibration parameters for POS, PSO, and SPO using fragmentation losses of ketenes TAG 2nd Precursor Ion observed Slope SD r 2 m/z Loss m/z Loss POS P O-ketene S-ketene O P-ketene S-ketene S P-ketene O-ketene PSO P O-ketene S-ketene O P-ketene S-ketene S P-ketene O-ketene SPO P O-ketene S-ketene O P-ketene S-ketene S P-ketene O-ketene does the same for the ketene-loss ions. The values of the slopes are for plots of concentration (mol L 1 ) on the X-axis versus the ratio of the ion counts of the calibrating TAG to the ion counts of the internal standard (OOS) on the Y-axis. The calibration curve slopes (sensitivities) confirm what has already been assumed the lactone-loss data show a higher sensitivity to regioisomerism in that the range in slopes is greater than for ketene-loss data and the slopes of the favored losses are higher for the lactone-loss data than

8 330 J Am Oil Chem Soc (2015) 92: for the ketene losses. With two exceptions the linearity of the calibration curves and the standard deviations of the slopes are quite satisfactory. These involve the loss of the O-lactone and P-lactone from the sn-1/3 positions of PSO, a very unfavorable loss with appropriate low values of the calibration slope. Since calibration is done in the presence of the fish oil matrix, all three TAG regioisomers will be present leading to a high background compared to the small intensity changes due to the PSO calibrant. Thus the signal-to-noise ratio is particularly low in these cases. It is probable that, if calibration could be accomplished without the fish oil matrix, these two calibrations would provide more satisfactory results. Quantification As mentioned above a TAG of type AAB requires two equations with four slopes to determine the concentrations of AAB and ABA. For PPO and POP these two equations could be taken as Set 1: I 583/329 = [PPO] [POP] and I 583/303 = [PPO] [POP] where I 583/329 is the intensity ratio of the m/z peak in the MS 3 spectrum with m/z as second-generation precursor ion and and are the slopes of the calibration curves of PPO and POP respectively measured under the same circumstances. Likewise, I 583/303 is the intensity ratio of the m/z peak with an m/z secondgeneration precursor ion and and are the corresponding slopes. However, six slopes are available (see Table 1), so the system is over-determined, which leads to the possibility of two other sets of simultaneous equations: Set 2: I 583/303 = [PPO] [POP] and I 557/303 = [PPO] [POP] and Set 3: I 583/329 = [PPO] [POP] and I 557/303 = [PPO] [POP] A type ABC TAG requires three equations with nine slopes to determine the concentrations of ABC, ACB, and BAC, yet there are eighteen slopes available (see Tables 2, 3). Again the system is over-determined and in this case there are twenty possible combinations for the simultaneous equations. It is probable that some of these equation sets will provide better results than others; the problem arises as to how to make the proper choice. Intuitively, a set of equations might be chosen with large differences in the slopes which vary in opposite directions in the equations. Any of the above three sets of equations would prove satisfactory under these conditions. A more rigorous mathematical approach would be to calculate the condition number of the slope matrix, CN = S S 1 where S is the slope matrix and S is the matrix norm. These terms are explained in greater detail in the supplementary material. The greater the condition number, the more ill-conditioned the matrix and the greater the possibility of error. Condition numbers do not decrease below unity. Calculations involving condition numbers are easily accomplished in a regular spreadsheet program. The condition numbers for the slope matrices of the three sets of equations above are 1.37, 2.77 and 5.69 respectively. In this study all three possible combinations of equations for PPO/POP were examined and two of the possible twenty combinations for POS/PSO/SPO were examined. These two combinations employed all eighteen slopes with no slopes in common and with large variations in the matrix coefficients. The sets of equations used are listed in the supplementary material. Correction for Isotope Pattern Influences Two types of corrections for isotope pattern effects, sometimes called 13 C isotope corrections, are often applied in lipid analysis [8, 11, 15, 35, 36]. The first type originates from the observation that the percentage of area of the [A+0] + peak, the one usually selected for analysis, decreases relative to other peaks in the isotope cluster as molecular mass increases. If a single standard is used to determine the response factor, assumed to be the same for all analytes, any difference in percentage area of the standard and analyte peak must be corrected. This type of correction is not necessary in this work since the analytes and standards are the same. Certain TAG also interfere in the analysis of selected analyte TAG. To avoid confusion the interfering TAG is designated as Int and the analyte TAG as Ana in the ions below. The second type of correction is needed because the [Ana+ 7 Li] + peak of the analyte overlaps the [Int+2+ 7 Li] + peak of a TAG interferent with the same ACN and one more double bond. Since these two molecules are not isomers, the possibility exists of separation by high resolution mass spectrometry. For example, in the case of POS, C 55 H 104 O 7 6 Li +, the [Ana+ 7 Li] + peak has m/z of and for the interferent [Int+2+ 7 Li] + ion, 12 C C 2 H 102 O 7 6 Li +, the m/z is A mass

9 J Am Oil Chem Soc (2015) 92: Fig. 2 MS 3 spectrum of the fish oil matrix: first-generation precursor ion m/z 867.8; second-generation precursor ion m/z (loss of 18:1); m/z = [16:0+ 7 Li] +, m/z = [18:0+ 7 Li] +, m/z = [A+ 7 Li 18:1 18:0- lactone] +, m/z = [A+ 7 Li 18:1 16:0-lactone] + spectrometer with a resolving power of at least 100,000 would be needed to separate these ions. Since the interfering ion contains heavy isotopes other than 13 C, its peak will be somewhat broader and require somewhat higher resolution. The [Int Li] + ion of a TAG with two more double bonds than the analyte will interfere in the same manner, but this interference is usually quite small and will not be considered here. In single-stage MS, all TAG with the same ACN and one double bond more than the analyte interfere. The use of MS 3 removes the interference mentioned above where two isomeric TAG have one acyl side chain in common. It also removes all the [Int+2+ 7 Li] + type interference unless the interfering TAG has two acyl side chains in common with the analyte, for example with POS, 16:0/18:1/18:0, the [Int+2+ 7 Li] + ion of 16:1/18:1/18:0 would interfere. All [Int+2+ 7 Li] + ions of TAG with the same ACN and one more DBN will be selected for fragmentation in the first stage of CID, but only those with two acid side chains in common with the analyte will give peaks in the MS 3 spectrum that overlap and interfere. If the second-generation precursor ion of the interferent is that which has lost the acid side chain with the extra double bond (16:1 in the example above), a single peak in the MS 3 spectrum results which coincides with an analyte peak and no correction is possible. If the second-generation precursor ion of the interferent has lost one of the common acid side chains (e.g., 18:1 or 18:0), an isotope pattern of three peaks appears in the MS 3 spectrum, two peaks at odd masses and one peak at an even mass. One of the odd-mass peaks will overlap the analyte peak. Evenmass peaks only appear in the MS 3 spectra of lithiated TAG due to this type of interference. The intensity of the even-mass peak and the isotope pattern [37] of the interferent can be used to correct for the interfering overlap. This type of interference is discussed in greater detail in the supplementary material. For a situation where the analyte and interferent have the same concentration, assuming equal ionization and fragmentation efficiency, fractional abundance calculations [37] indicate that this type of interference will result in about a 20 % error in peak area for single-stage MS, an error of between 2 and 10 % for MS 2 and an error of about 2 % for MS 3 for a TAG such as POS. Figure 2 illustrates the presence of even mass peaks in the MS 3 spectrum of the fish oil indicating this type of interference. If natural lithium with approximately 7 % 6 Li were used as cationization reagent, the [Ana+1+ 6 Li] + TAG peaks, which contain mainly the heavier lithium-7 isotope, would be selected for analysis for sensitivity considerations. The resulting spectra would contain even-mass peaks due to the inclusion of some 6 Li in the ions, making the use of even-mass peaks for correction as described above much more difficult. This situation is avoided through the use of isotopically pure 7 Li. It should be noted that interference from [Int+2+ 7 Li] + ions does not affect the slopes of the calibration curves, only the intercepts. Thus calibration can be performed without correction for this interference. This was verified experimentally. Software, written in-house, was used to identify even mass peaks adjacent to the analyte peaks of interest and make corrections based on an algorithm that approximated the appropriate isotope pattern. This can also be accomplished with spreadsheet programs as discussed in more detail in the supplementary material. Determination of Background Levels in the Fish Oil Matrix Using the lactone-loss peak intensities and slopes, the set of equations with the lowest slope-matrix condition number (Set 1) provided the results that the concentration of PPO in the fish oil was M (0.45 % by weight in the oil) and that of POP was M (0.081 %). Averaging the data from all three sets of equations gave M (0.44 %) and M (0.067 %) respectively. Using

10 332 J Am Oil Chem Soc (2015) 92: the ketene loss data set with the best condition number (Set 1, see the supplementary material) gave M (0.48 %) and M (0.12 %) for PPO and POP, while the average of all three sets of equations gave M (0.49 %) and M (0.083 %), respectively. The agreement among these data sets is satisfactory at these concentration levels. The sets of equations used for the analysis of POS, PSO, and SPO had almost identical condition numbers. The lactone-loss data provided the following results: [POS] = M (0.091 %), [PSO] = M (0.026 %), [SPO] = M (0.26 %) for equations Set 1 and [POS] = M (0.075 %), [PSO] = M (0.024 %) and [SPO] = M (0.33 %) for equations Set 2. The ketene-loss data produced the following: [POS] = M (0.12 %), [PSO] = 0 M (0 %), [SPO] = M (0.60 %) for equations Set 1 and [POS] = M (0.35 %), [PSO] = 0 M (0 %) and [SPO] = M (0.35 %) for equations Set 2. The values for [PSO] from the keteneloss data were actually small negative numbers which were set to zero. The lactone-loss data indicate that the concentration of POS in the fish oil is quite small. The average difference in concentration values for the two equation sets used for the lactone-loss data is about 14 %. Since this analysis involved minor components in a complex sample at very low concentrations, to M, this level of agreement is reasonable. The ketene-loss data provided higher values, except for PSO, than the lactone-loss data and the agreement between equation sets was not as good. Even so, the ketene-loss data and lactone-loss data gave the same order in concentrations, [SPO] > [POS] > [PSO]. As stated above the ketene-loss data are not expected to provide results as good as the lactone-loss data. The ketene-loss data set for POS/PSO/SPO lacked intensity and had poor precision, even compared to the ketene-loss set for PPO/POP, which is a probable cause for the disagreement. Malone and Evans [38] found that palmitic acid was detected almost exclusively in the sn-2 position in pork fat. The results above indicate that palmitic acid is also found preferentially in this position in those TAG examined in the fish oil sample used in this study. Validation of the Method with the Spiked Synthetic Samples Various concentrations of the analyte TAG were added to the fish oil and the total analyte concentrations (or expected analyte concentrations) were calculated as the sum of the added concentrations plus the background concentrations. The percentage error was determined from the difference between the expected and experimentally measured concentrations. The background and experimentally measured concentrations were calculated using the same slope data. An example of a set of synthetic samples for PPO and POP is given in Table S3 in the supplementary material. Tables 4 and 5 list the average percentage errors calculated for the synthetic samples. As expected, the errors are smaller when the lactone-loss data rather than the keteneloss data are used in the calculations, especially for the POS/PSO/SPO samples. The ketene-loss data for equation Set 2 for the PPO/POP samples are particularly errorprone. The slope-matrix for this equation set has a particularly high condition number, 15.7, while none of the other condition numbers exceeded 5.7 and most were less than 3.4. Correcting for interference from the [Int+2+ 7 Li] + peaks of TAG with the same ACN and one more double bond did not significantly change the average percentage error values, but it did lower the calculated concentrations, as expected. For the samples with higher concentrations (~ M), the change in concentration after correction averaged about 0.5 percent while for more dilute samples (~ M) the change averaged more than three percent. Table 4 Average percentage error values for the nine synthetic samples containing PPO and POP Data Equation set 1 Equation set 2 Equation set 3 PPO POP PPO POP PPO POP Lactone-loss Ketene-loss Table 5 Average percentage error values for the twelve synthetic samples containing POS, PSO, and SPO Data Equation set 1 Equation set 2 POS PSO SPO POS PSO SPO Lactone-loss Ketene-loss

11 J Am Oil Chem Soc (2015) 92: Conclusions The use of ESI to form lithium adducts of TAG in complex oil matrices followed by triple-stage mass spectrometry can provide both absolute and relative quantitative data for regioisomers of both AAB and ABC type TAG with average errors in the range of five percent. This can be accomplished by simply dissolving the oil in an appropriate solvent (methanol), adding a low concentration of a lithium-7 salt and an internal standard and performing an infusion MS analysis; no chromatography is necessary. The peaks in the MS 3 spectra that arise from the loss of a lactone would be used as the data source, not those arising from a ketene loss. However, the ability of the ketene-loss peaks and the different equation sets to provide useful information emphasizes the utility of the method. An equation set with a low slope-matrix condition number should be employed in the analysis. This method avoids interference from isomeric TAG with one acyl side chain in common and from all TAG with the same ACN but with one more double bond except for interferents with two acyl side chains in common. In this case isotope cluster corrections can be applied for the overlap in peaks when the second-generation precursor ion of the interferent has lost the same acyl chain as the analyte. Corrections cannot be applied to the situation where the interferent loses the acyl side chain with one more double bond than the analyte. The need for corrections will depend on the concentrations of the analyte and interferent and will be sample-dependent. For analytes with a high concentration, there will probably be little need for correction. The advantages of the method are that it can be performed quickly without prior chromatography and with a minimum number of steps and is not time-limited in that the sample can be infused for as long as needed with no change in solvent or matrix. The main disadvantage is that it requires appropriate pure TAG as standards. This does, however, avoid the need to make assumptions about response factors and make corrections for changes in isotope pattern with molecular mass. A less serious disadvantage would be the need to correct for [Int+2+ 7 Li] + interference with some samples and the inability to make such corrections under certain circumstances. Finally it has the disadvantage of providing no data on the position or geometry of double bonds in the acyl side chains. However, Hsu and Turk [20] have suggested that some charge remote fragmentations in the spectra of lithiated TAG might be useful in this regard. Calibration must be done in the presence of the matrix, which may present problems in the determination of the slopes of minor peaks. This method would not be used for the exhaustive analysis of an oil, unless that oil were very simple, since each TAG has to be examined individually, requiring 333 preknowledge of the sample composition. It is well suited for routine targeted analysis of a group of TAG in a number of well-characterized samples of varying complexity. Under these conditions the effort and expense of providing pure standards for the TAG involved may be justified. Holčapek et al. [2] have proposed a method of preparing various standards by randomization from TAG of type AAA, which are relatively easily obtained. Acknowledgments The authors wish to acknowledge the helpful discussions with Prof. J. S. Grossert during the preparation of this manuscript. References 1. Leskinen H, Suomela J-P, Kallio H (2007) Quantification of triacylglycerol regioisomers in oils and fat using different mass spectrometric and liquid chromatographic methods. 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