Journal of Chromatography A, 1360 (2014) 172–187
Contents lists available at ScienceDirect
Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
High performance characterization of triacylglycerols in milk and milk-related samples by liquid chromatography and mass spectrometry夽 Marco Beccaria a , Giuseppe Sullini a , Francesco Cacciola b,c , Paola Donato a,c,d , Paola Dugo a,c,d , Luigi Mondello a,c,d,∗ a
Dipartimento di Scienze del Farmaco e dei Prodotti per la Salute (S.C.I.F.A.R.), University of Messina, Viale Annunziata, 98168 Messina, Italy Dipartimento di Scienze dell’Ambiente, della Sicurezza, del Territorio, degli Alimenti e della Salute (S.A.S.T.A.S.), University of Messina, Viale F. Stagno d’Alcontres 31, 98166 Messina, Italy c Chromaleont s.r.l. A start-up of the University of Messina, c/o Dipartimento di Scienze del Farmaco e dei Prodotti per la Salute (S.C.I.F.A.R.), University of Messina, Viale Annunziata, 98168 Messina, Italy d Centro Integrato di Ricerca (C.I.R.), University Campus Bio-Medico of Rome, Via Álvaro del Portillo 21, 00128 Rome, Italy b
a r t i c l e
i n f o
Article history: Received 12 December 2013 Received in revised form 26 June 2014 Accepted 23 July 2014 Available online 1 August 2014 Keywords: Milk Triacylglycerols Mass spectrometry Serially-coupled columns Fused-core
a b s t r a c t In this work, ultra high performance liquid chromatography was used for the characterization of non polar lipids (triacylglycerols) in milk samples of different origin, as well as milk-derivatives. For tackling such a task, three core–shell type octadecylsilica columns were serially coupled, reaching a total stationary phase length of 45 cm, using acetonitrile-isopropanol gradient elution allowing triacylglicerol separation according to increasing partition number. The employment of an ion-trap-time-of-flight detection in conjunction with atmospheric-pressure chemical ionization mass spectrometry was carried out to positively identify a number of 243 different triacylglycerols containing up to 22 fatty acids, with 2–22 carbon atom alkyl chain length, and 0–3 double bonds. This work reports an extensive characterization of the triacylglycerol fraction in milk and milk-related samples of different sources. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Nowadays there is a great deal of interest in the composition of triacylglycerols (TAGs) in dietary fats due to their influence on physiology and nutritional aspects. Milk fat is a very complex source of TAGs, and several factors such as climate, diet, stage of lactation may strongly influence the chemical composition [1]. In general, the extremely high number of fatty acids (FAs) that can be esterified to the three hydroxyl groups of the glycerol backbone, differing in chain length and number of double bonds (DBs), gives rise to a large number of TAG mixtures.
夽 Presented at the 20th International Symposium on Electro- and Liquid PhaseSeparaton Techniques (ITP 2013), 6–9 October 2013, Puerto de la Cruz, Tenerife, Canary Islands, Spain. ∗ Corresponding author at: Corresponding author. Tel.: +39 090 6766536; fax: +39 090 358220. E-mail address: [email protected] (L. Mondello). http://dx.doi.org/10.1016/j.chroma.2014.07.073 0021-9673/© 2014 Elsevier B.V. All rights reserved.
For TAG characterization, both reversed-phase liquid chromatography (RPLC) and silver-ion chromatography (SIC) methods have been employed [2–23]. In SIC, separation occurs on the basis of unsaturation degree, as well as on DB position or configuration in each FA [3–5]; under RPLC conditions, TAG separation occurs on the basis of the partition number (PN), which is equal to the carbon number of the three FAs minus twice the number of DBs (PN = CN-2DBs). The latter technique, performed under non aqueous reversed-phase liquid chromatography conditions (NARP), is by far the most employed technique for attaining a detailed information on TAG composition in natural samples, as witnessed by the several research works on this field [6–25]. Concerning detection, both UV and evaporative light-scattering (ELS) detection have been employed for TAG analysis [6,10,13], even though mass spectrometry (MS) in combination with LC has gained an ever increasing interest for lipid determination [5–9,11,12,14–28]. Knowledge of the composition of milk lipids, in terms of TAGs, has been evaluated in order to improve the quality of infant-fed formula, as most clinical studies have shown e.g. human milk is
M. Beccaria et al. / J. Chromatogr. A 1360 (2014) 172–187
better absorbed, particularly in premature and newborn infants, due to its TAG composition [29,30]. Lipid extraction is a critical step in the analysis of total lipids, since contamination or improper extraction of components of interest may occur leading to misinterpretations [31]. During lipid extraction, samples should be prepared and analyzed carefully to prevent oxidation of lipids and hydrolysis, since artefact production can compromise the identification and quantification of the lipid fraction components [32]. A number of works have already dealt with such a topic, however an extensive milk TAG characterization is missing [14–27]. This is partially related to the very high complexity of lipidic mixtures, often exceeding the separation capability afforded by any single column. Two approaches have been developed so far, to face such an issue: the first consisted in the use of serially-coupled columns [19,20,22,28], the second in the implementation of multidimensional LC systems (MD-LC) for TAG characterization [24,25]. In this contribution, a characterization and comparison of an optimized NARP-UHPLC (ultra high pressure liquid chromatography) method, in combination with positive atmospheric-pressure chemical ionization ion trap-time of flight-mass spectrometry (APCI-IT-TOF-MS) detection was developed for TAG separation in milk and milk-derivatives of different origin. This is the only example reported in literature by using an UHPLC–MS technique for TAG analysis in milk samples and milk-derivates with core–shell type C18 columns. Aiming to estimate the gain in efficiency under true UHPLC operating pressures, the performance of the gradient separation, in terms of peak capacity (nC ), by switching from a single C18 to two- and three-serially coupled C18 columns was evaluated by using a standard mixture of six different TAGs spanning over a broad PN range. Furthermore, thanks to the employment of the IT-TOF detection, accurate mass calculations were reported for all identified TAGs in the most complex sample viz. goat milk. 2. Experimental 2.1. Samples and chemicals Acetonitrile (ACN) and isopropanol (IPA), both MS grade, were obtained from Sigma–Aldrich (Milan, Italy). The tested samples were attained as follows: goat and cow milk from the Calabria region, human milk from a sane voluntary, buffalo mozzarella cheese from the Campania region. 2.2. Sample preparation Exhaustive extraction of the whole lipid content for the milk samples was achieved according to the Folch extraction method [33], whereas the buffalo mozzarella cheese was treated with the Schmid–Bondzynski–Ratzlaff (SBR) method [34]. The extraction of the milk lipid fraction was carried out from 10 mL of each sample using 40 mL of a chloroform/methanol (2:1, v/v) mixture. The sample/solvent mixture was agitated thoroughly using a vortex for 30 min in an ice bath. Afterwards, the resulting content was left to stand for 5 min, and later on centrifuged in a centrifuge tube (15 min). Once the centrifugation step was completed, the lower phase was carefully taken. The aqueous phase was reextracted with 20 mL of a chloroform/methanol (2:1, v/v) mixture for two times. The total extract was dried over anhydrous sodium sulfate, filtered and evaporated under vacuum; the final dry residue was stored at −18 ◦ C until use. The extraction of the buffalo mozzarella cheese lipid fraction was accomplished from 10 g of the sample accurately weighed and dissolved in 20 ml of hydrochloric acid and 20 ml of ethyl alcohol.
173
The preparation was done in a volumetric flask inserted in a boiling water-bath and kept gently moved (for 30 min at 50 ◦ C) with constant magnetic stirring until complete dissolution. Afterwards, the flask was cooled down in running water and a mixture of n-hexane and ethyl ether (1:2, v/v) was added and the mixture was shaked for additional 15 min. The suspension was let to stand for 10 min to allow phase separation. The extraction protocol was repeated 3 times. The organic extracts were pooled, dried over anhydrous sodium sulfate, filtered and then brought to dryness under vacuum; the final dry residue was stored at −18 ◦ C until use. All the samples were weighted in a range from 40 to 50 mg, diluted with 10 mL of acetone and afterwards filtered through a 0.22 m Acrodisc nylon membrane filter (Pall Life Sciences, Ann Arbor, MI, USA) prior to LC–MS analyses. 2.3. (NARP)LC/APCI-MS analyses Analyses were carried out on a Nexera liquid chromatography system (Shimadzu, Milan, Italy), already described in Ref. [28], coupled to an LCMS-IT-TOF mass spectrometer through an APCI source (Shimadzu, Kyoto, Japan). Data acquisition was performed by means of the LCMSsolution software (Version 3.50.346, Shimadzu). Chromatographic separation was achieved on Ascentis Express Fused-core C18 columns, 150 mm × 4.6 mm i.d., 2.7 m d.p., kindly donated by Supelco/Sigma–Aldrich (Bellefonte, PA, USA). Mobile phases employed consisted of: (A) ACN, and (B) IPA, under the following gradient: 0 min, 0% B; 50 min, 70% B (hold for 5 min); 56 min, 0% B. The mobile phase flow rate was 1 mL/min. When switching from one- to multi-column set-up, the gradient was adapted on the coupled columns accordingly, with the xL factor; i.e. for the three-column configuration, each gradient step was three-fold increased, viz.: 0 min, 0% B; 150 min, 70% B (hold for 15 min); 168 min, 0% B. LCMS-IT-TOF detection was achieved through an APCI interface operated in positive ionization mode under the following conditions: detector voltage, 1.50 kV; interface temperature: 400 ◦ C; CDL temperature, 230 ◦ C; block heater temperature, 230 ◦ C; nebulizing gas flow (N2 ), 2.0 L/min; drying gas flow (N2 ), 15.0 L/min; ion accumulation time, 50 ms; full scan range, 200–1200 m/z; event time, 300 ms; repeat, 3; ASC, 70%. Resolving power (FWHM defined at m/z): 10,000 (m/z 1000); Resolution (m/z): 0.1. 2.4. GC–FID and GC–MS analyses Instrumentation and analytical conditions employed for the buffalo mozzarella cheese sample are the same already reported in Ref. [44] 3. Results and discussion 3.1. Evaluation of column performance on serially-coupled Ascentis Express C18 columns for triacylglycerol analysis As a starting point, the different milk samples were analyzed on an Ascentis Express C18 column, with a linear gradient of increasing IPA in ACN in a run time of roughly 55 min. Such a stationary phase has already been successfully investigated in a number of research works coming from the same and other research groups [12,13,28,35–42]. Fig. 1 shows the (NARP)LC/APCIMS chromatograms of the analyzed TAGs on the goat (A), cow (B), human (C) milk and mozzarella cheese (D) samples analyzed on an Ascentis Express C18 column. From a visual inspection it can be appreciated that the goat milk was the most complex one among the Folch milk samples investigated. TAGs were eluted according to increasing PN, reflecting the number of CNs and DBs in acyl
174
M. Beccaria et al. / J. Chromatogr. A 1360 (2014) 172–187
Fig. 1. TIC (total ion current) chromatograms of the (NARP)LC/APCI-MS for the goat (A), cow (B), human (C) milk and mozzarella cheese (D) samples analyzed on an Ascentis Express C18 column. For experimental conditions, see the text.
chains. Despite a partial separation was achieved, identification of the majority of the components was extremely complicated due to the co-elutions, especially for intermediate PN TAG species. Under such conditions, a correct interpretation of mass spectra is hampered since an ion at a particular mass corresponds to a number of different diacylglycerol fragments as recently illustrated for a very complex fish oil sample [28]. To overcome such an issue, aiming to increase the overall resolving power, three fused core stationary phases were serially-coupled reaching a total length of 45 cm and operating under UHPLC conditions. A flow rate of 1 mL/min gave the best results, in terms of peak overlap and resolution, allowing
Fig. 2. TIC (total ion current) chromatograms of the (NARP)LC/APCI-MS for the same samples reported in Fig. 1 on three serially coupled Ascentis Express C18 columns. For experimental conditions, see the text.
a straightforward hyphenation to the APCI-MS detection [28]. To keep the same retention factors and to have comparable sensitivity, the gradient program and the injected volumes were adapted on the coupled columns by an increase of two- and three-fold. The resulting chromatograms of the various milk and buffalo mozzarella cheese samples tested are reported in Fig. 2. As expected, a much broader PN range, ranging from 22 to 54, was observed for the goat and cow milk and mozzarella cheese samples, differently from the human milk sample (PN range from 38 to 52), due to the lack of short fatty acids.
M. Beccaria et al. / J. Chromatogr. A 1360 (2014) 172–187
175
Table 1 Peak capacity values for a TAG standard mixture on one and two- and three serially coupled C18 columns. Chromatographic set-up
tg
n
Wtotal (min)
nC
Theor. Rs
%RSD tR
Backpressure at 70% IPA (MPa)
One column (15 cm) Two serially-coupled columns (30 cm) Three serially-coupled columns (45 cm)
38 76 114
5 5 5
0.37 0.58 0.68
104 133 170
1.0 1.39 1.72
0.60 0.58 1.09
20.5 40.5 59.5
In order to evaluate the performance of the gradient separation, by switching a single C18 to two- and three-serially coupled C18 columns, the peak capacity (nC ) was calculated using the method defined by Neue [43]: nC = 1 +
tg (1/n)
n 1
w
in which tg is the gradient time, n is the number of peaks selected for the calculation, and w the average peak width. For such a calculation, a standard mixture of six TAGs, with a broad number of PN, viz. from 24 to 48, composed of C8:0C8:0C8:0 (tricaprilin), C10:0C10:0C10:0 (tricaprin), C12:0C12:0C12:0 (trilaurin), C14:0C14:0C14:0 (trimiristin), C18:1C18:1C18:1 (triolein), C16:0C16:0C16:0 (tripalmitin) was analyzed under the same experimental conditions used for the different samples investigated. The results attained are reported in Table 1. nC increased from 104 on the single C18 column to 170 for the three serially coupled C18 columns. The experimentally calculated nC ratios were in good accordance with the theoretical gain in resolution, calculated for single pairs of TAG tested, namely C8:0C8:0C8:0 and C10:0C10:0C10:0, C10:0C10:0C10:0 and C12:0C12:0C12:0, C12:0C12:0C12:0 and C14:0C14:0C14:0, C14:0C14:0C14:0 and C18:1C18:1C18:1, C18:1C18:1C18:1 and C16:0C16:0C16:0; values of 1.39 and 1.72 were measured, when doubling and tripling the length of stationary phases, respectively. Repeatibility of retention times was calculated on five replicates in each set-up tested with RSD% values ranging from 0.60 for the single C18 column to 0.58 and 1.09 for the two- and three-serially coupled columns, respectively (Table 1). The experimental pressure drop, to 70% IPA in ACN, was 20.5 MPa for the single C18 column to 59.5 MPa for the three serially coupled C18 columns. The set-up comprised of three serially coupled columns yielded backpressure values not compatible with a conventional HPLC instrument thus requiring the employment of ultra high pressure pumps. Being feasible, the addition of a fourth column was also tested but afterwards neglected compromising between resolution and retention times (data not shown). 3.2. Elucidation of the triacylglycerol composition in milk samples by (NARP)LC/APCI-MS Prior to the characterization of the triacylglycerol composition of the various milk and milk-derivative samples tested, GC–FID (gas chromatography–flame ionization detection) data were considered. For such a task, the results recently reported on the same goat, cow and human milk samples were used [44] whereas the fatty acid content of the buffalo mozzarella cheese sample is illustrated in Table S1. A complete list of FAs occurring in the identified TAGs investigated in this work is reported in Table 2. In all the samples investigated, the highest percentage of total FAs was represented by saturated FAs, followed by monounsaturated FAs and afterwards by polyunsaturated FAs. In particular the goat and cow milk samples contained the highest percentage of saturated FAs (roughly 74%), while the human milk sample only 46% of saturated FAs, and a nearly equal content of the monounsaturated ones (43%). The buffalo mozzarella cheese extract was similar to the goat and
cow milk samples with a percentage of saturated FAs roughly 70% followed by monounsaturated FAs. The relative abundance (%) of the protonated [M+H]+ ions can be very low (
Contents lists available at ScienceDirect
Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
High performance characterization of triacylglycerols in milk and milk-related samples by liquid chromatography and mass spectrometry夽 Marco Beccaria a , Giuseppe Sullini a , Francesco Cacciola b,c , Paola Donato a,c,d , Paola Dugo a,c,d , Luigi Mondello a,c,d,∗ a
Dipartimento di Scienze del Farmaco e dei Prodotti per la Salute (S.C.I.F.A.R.), University of Messina, Viale Annunziata, 98168 Messina, Italy Dipartimento di Scienze dell’Ambiente, della Sicurezza, del Territorio, degli Alimenti e della Salute (S.A.S.T.A.S.), University of Messina, Viale F. Stagno d’Alcontres 31, 98166 Messina, Italy c Chromaleont s.r.l. A start-up of the University of Messina, c/o Dipartimento di Scienze del Farmaco e dei Prodotti per la Salute (S.C.I.F.A.R.), University of Messina, Viale Annunziata, 98168 Messina, Italy d Centro Integrato di Ricerca (C.I.R.), University Campus Bio-Medico of Rome, Via Álvaro del Portillo 21, 00128 Rome, Italy b
a r t i c l e
i n f o
Article history: Received 12 December 2013 Received in revised form 26 June 2014 Accepted 23 July 2014 Available online 1 August 2014 Keywords: Milk Triacylglycerols Mass spectrometry Serially-coupled columns Fused-core
a b s t r a c t In this work, ultra high performance liquid chromatography was used for the characterization of non polar lipids (triacylglycerols) in milk samples of different origin, as well as milk-derivatives. For tackling such a task, three core–shell type octadecylsilica columns were serially coupled, reaching a total stationary phase length of 45 cm, using acetonitrile-isopropanol gradient elution allowing triacylglicerol separation according to increasing partition number. The employment of an ion-trap-time-of-flight detection in conjunction with atmospheric-pressure chemical ionization mass spectrometry was carried out to positively identify a number of 243 different triacylglycerols containing up to 22 fatty acids, with 2–22 carbon atom alkyl chain length, and 0–3 double bonds. This work reports an extensive characterization of the triacylglycerol fraction in milk and milk-related samples of different sources. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Nowadays there is a great deal of interest in the composition of triacylglycerols (TAGs) in dietary fats due to their influence on physiology and nutritional aspects. Milk fat is a very complex source of TAGs, and several factors such as climate, diet, stage of lactation may strongly influence the chemical composition [1]. In general, the extremely high number of fatty acids (FAs) that can be esterified to the three hydroxyl groups of the glycerol backbone, differing in chain length and number of double bonds (DBs), gives rise to a large number of TAG mixtures.
夽 Presented at the 20th International Symposium on Electro- and Liquid PhaseSeparaton Techniques (ITP 2013), 6–9 October 2013, Puerto de la Cruz, Tenerife, Canary Islands, Spain. ∗ Corresponding author at: Corresponding author. Tel.: +39 090 6766536; fax: +39 090 358220. E-mail address: [email protected] (L. Mondello). http://dx.doi.org/10.1016/j.chroma.2014.07.073 0021-9673/© 2014 Elsevier B.V. All rights reserved.
For TAG characterization, both reversed-phase liquid chromatography (RPLC) and silver-ion chromatography (SIC) methods have been employed [2–23]. In SIC, separation occurs on the basis of unsaturation degree, as well as on DB position or configuration in each FA [3–5]; under RPLC conditions, TAG separation occurs on the basis of the partition number (PN), which is equal to the carbon number of the three FAs minus twice the number of DBs (PN = CN-2DBs). The latter technique, performed under non aqueous reversed-phase liquid chromatography conditions (NARP), is by far the most employed technique for attaining a detailed information on TAG composition in natural samples, as witnessed by the several research works on this field [6–25]. Concerning detection, both UV and evaporative light-scattering (ELS) detection have been employed for TAG analysis [6,10,13], even though mass spectrometry (MS) in combination with LC has gained an ever increasing interest for lipid determination [5–9,11,12,14–28]. Knowledge of the composition of milk lipids, in terms of TAGs, has been evaluated in order to improve the quality of infant-fed formula, as most clinical studies have shown e.g. human milk is
M. Beccaria et al. / J. Chromatogr. A 1360 (2014) 172–187
better absorbed, particularly in premature and newborn infants, due to its TAG composition [29,30]. Lipid extraction is a critical step in the analysis of total lipids, since contamination or improper extraction of components of interest may occur leading to misinterpretations [31]. During lipid extraction, samples should be prepared and analyzed carefully to prevent oxidation of lipids and hydrolysis, since artefact production can compromise the identification and quantification of the lipid fraction components [32]. A number of works have already dealt with such a topic, however an extensive milk TAG characterization is missing [14–27]. This is partially related to the very high complexity of lipidic mixtures, often exceeding the separation capability afforded by any single column. Two approaches have been developed so far, to face such an issue: the first consisted in the use of serially-coupled columns [19,20,22,28], the second in the implementation of multidimensional LC systems (MD-LC) for TAG characterization [24,25]. In this contribution, a characterization and comparison of an optimized NARP-UHPLC (ultra high pressure liquid chromatography) method, in combination with positive atmospheric-pressure chemical ionization ion trap-time of flight-mass spectrometry (APCI-IT-TOF-MS) detection was developed for TAG separation in milk and milk-derivatives of different origin. This is the only example reported in literature by using an UHPLC–MS technique for TAG analysis in milk samples and milk-derivates with core–shell type C18 columns. Aiming to estimate the gain in efficiency under true UHPLC operating pressures, the performance of the gradient separation, in terms of peak capacity (nC ), by switching from a single C18 to two- and three-serially coupled C18 columns was evaluated by using a standard mixture of six different TAGs spanning over a broad PN range. Furthermore, thanks to the employment of the IT-TOF detection, accurate mass calculations were reported for all identified TAGs in the most complex sample viz. goat milk. 2. Experimental 2.1. Samples and chemicals Acetonitrile (ACN) and isopropanol (IPA), both MS grade, were obtained from Sigma–Aldrich (Milan, Italy). The tested samples were attained as follows: goat and cow milk from the Calabria region, human milk from a sane voluntary, buffalo mozzarella cheese from the Campania region. 2.2. Sample preparation Exhaustive extraction of the whole lipid content for the milk samples was achieved according to the Folch extraction method [33], whereas the buffalo mozzarella cheese was treated with the Schmid–Bondzynski–Ratzlaff (SBR) method [34]. The extraction of the milk lipid fraction was carried out from 10 mL of each sample using 40 mL of a chloroform/methanol (2:1, v/v) mixture. The sample/solvent mixture was agitated thoroughly using a vortex for 30 min in an ice bath. Afterwards, the resulting content was left to stand for 5 min, and later on centrifuged in a centrifuge tube (15 min). Once the centrifugation step was completed, the lower phase was carefully taken. The aqueous phase was reextracted with 20 mL of a chloroform/methanol (2:1, v/v) mixture for two times. The total extract was dried over anhydrous sodium sulfate, filtered and evaporated under vacuum; the final dry residue was stored at −18 ◦ C until use. The extraction of the buffalo mozzarella cheese lipid fraction was accomplished from 10 g of the sample accurately weighed and dissolved in 20 ml of hydrochloric acid and 20 ml of ethyl alcohol.
173
The preparation was done in a volumetric flask inserted in a boiling water-bath and kept gently moved (for 30 min at 50 ◦ C) with constant magnetic stirring until complete dissolution. Afterwards, the flask was cooled down in running water and a mixture of n-hexane and ethyl ether (1:2, v/v) was added and the mixture was shaked for additional 15 min. The suspension was let to stand for 10 min to allow phase separation. The extraction protocol was repeated 3 times. The organic extracts were pooled, dried over anhydrous sodium sulfate, filtered and then brought to dryness under vacuum; the final dry residue was stored at −18 ◦ C until use. All the samples were weighted in a range from 40 to 50 mg, diluted with 10 mL of acetone and afterwards filtered through a 0.22 m Acrodisc nylon membrane filter (Pall Life Sciences, Ann Arbor, MI, USA) prior to LC–MS analyses. 2.3. (NARP)LC/APCI-MS analyses Analyses were carried out on a Nexera liquid chromatography system (Shimadzu, Milan, Italy), already described in Ref. [28], coupled to an LCMS-IT-TOF mass spectrometer through an APCI source (Shimadzu, Kyoto, Japan). Data acquisition was performed by means of the LCMSsolution software (Version 3.50.346, Shimadzu). Chromatographic separation was achieved on Ascentis Express Fused-core C18 columns, 150 mm × 4.6 mm i.d., 2.7 m d.p., kindly donated by Supelco/Sigma–Aldrich (Bellefonte, PA, USA). Mobile phases employed consisted of: (A) ACN, and (B) IPA, under the following gradient: 0 min, 0% B; 50 min, 70% B (hold for 5 min); 56 min, 0% B. The mobile phase flow rate was 1 mL/min. When switching from one- to multi-column set-up, the gradient was adapted on the coupled columns accordingly, with the xL factor; i.e. for the three-column configuration, each gradient step was three-fold increased, viz.: 0 min, 0% B; 150 min, 70% B (hold for 15 min); 168 min, 0% B. LCMS-IT-TOF detection was achieved through an APCI interface operated in positive ionization mode under the following conditions: detector voltage, 1.50 kV; interface temperature: 400 ◦ C; CDL temperature, 230 ◦ C; block heater temperature, 230 ◦ C; nebulizing gas flow (N2 ), 2.0 L/min; drying gas flow (N2 ), 15.0 L/min; ion accumulation time, 50 ms; full scan range, 200–1200 m/z; event time, 300 ms; repeat, 3; ASC, 70%. Resolving power (FWHM defined at m/z): 10,000 (m/z 1000); Resolution (m/z): 0.1. 2.4. GC–FID and GC–MS analyses Instrumentation and analytical conditions employed for the buffalo mozzarella cheese sample are the same already reported in Ref. [44] 3. Results and discussion 3.1. Evaluation of column performance on serially-coupled Ascentis Express C18 columns for triacylglycerol analysis As a starting point, the different milk samples were analyzed on an Ascentis Express C18 column, with a linear gradient of increasing IPA in ACN in a run time of roughly 55 min. Such a stationary phase has already been successfully investigated in a number of research works coming from the same and other research groups [12,13,28,35–42]. Fig. 1 shows the (NARP)LC/APCIMS chromatograms of the analyzed TAGs on the goat (A), cow (B), human (C) milk and mozzarella cheese (D) samples analyzed on an Ascentis Express C18 column. From a visual inspection it can be appreciated that the goat milk was the most complex one among the Folch milk samples investigated. TAGs were eluted according to increasing PN, reflecting the number of CNs and DBs in acyl
174
M. Beccaria et al. / J. Chromatogr. A 1360 (2014) 172–187
Fig. 1. TIC (total ion current) chromatograms of the (NARP)LC/APCI-MS for the goat (A), cow (B), human (C) milk and mozzarella cheese (D) samples analyzed on an Ascentis Express C18 column. For experimental conditions, see the text.
chains. Despite a partial separation was achieved, identification of the majority of the components was extremely complicated due to the co-elutions, especially for intermediate PN TAG species. Under such conditions, a correct interpretation of mass spectra is hampered since an ion at a particular mass corresponds to a number of different diacylglycerol fragments as recently illustrated for a very complex fish oil sample [28]. To overcome such an issue, aiming to increase the overall resolving power, three fused core stationary phases were serially-coupled reaching a total length of 45 cm and operating under UHPLC conditions. A flow rate of 1 mL/min gave the best results, in terms of peak overlap and resolution, allowing
Fig. 2. TIC (total ion current) chromatograms of the (NARP)LC/APCI-MS for the same samples reported in Fig. 1 on three serially coupled Ascentis Express C18 columns. For experimental conditions, see the text.
a straightforward hyphenation to the APCI-MS detection [28]. To keep the same retention factors and to have comparable sensitivity, the gradient program and the injected volumes were adapted on the coupled columns by an increase of two- and three-fold. The resulting chromatograms of the various milk and buffalo mozzarella cheese samples tested are reported in Fig. 2. As expected, a much broader PN range, ranging from 22 to 54, was observed for the goat and cow milk and mozzarella cheese samples, differently from the human milk sample (PN range from 38 to 52), due to the lack of short fatty acids.
M. Beccaria et al. / J. Chromatogr. A 1360 (2014) 172–187
175
Table 1 Peak capacity values for a TAG standard mixture on one and two- and three serially coupled C18 columns. Chromatographic set-up
tg
n
Wtotal (min)
nC
Theor. Rs
%RSD tR
Backpressure at 70% IPA (MPa)
One column (15 cm) Two serially-coupled columns (30 cm) Three serially-coupled columns (45 cm)
38 76 114
5 5 5
0.37 0.58 0.68
104 133 170
1.0 1.39 1.72
0.60 0.58 1.09
20.5 40.5 59.5
In order to evaluate the performance of the gradient separation, by switching a single C18 to two- and three-serially coupled C18 columns, the peak capacity (nC ) was calculated using the method defined by Neue [43]: nC = 1 +
tg (1/n)
n 1
w
in which tg is the gradient time, n is the number of peaks selected for the calculation, and w the average peak width. For such a calculation, a standard mixture of six TAGs, with a broad number of PN, viz. from 24 to 48, composed of C8:0C8:0C8:0 (tricaprilin), C10:0C10:0C10:0 (tricaprin), C12:0C12:0C12:0 (trilaurin), C14:0C14:0C14:0 (trimiristin), C18:1C18:1C18:1 (triolein), C16:0C16:0C16:0 (tripalmitin) was analyzed under the same experimental conditions used for the different samples investigated. The results attained are reported in Table 1. nC increased from 104 on the single C18 column to 170 for the three serially coupled C18 columns. The experimentally calculated nC ratios were in good accordance with the theoretical gain in resolution, calculated for single pairs of TAG tested, namely C8:0C8:0C8:0 and C10:0C10:0C10:0, C10:0C10:0C10:0 and C12:0C12:0C12:0, C12:0C12:0C12:0 and C14:0C14:0C14:0, C14:0C14:0C14:0 and C18:1C18:1C18:1, C18:1C18:1C18:1 and C16:0C16:0C16:0; values of 1.39 and 1.72 were measured, when doubling and tripling the length of stationary phases, respectively. Repeatibility of retention times was calculated on five replicates in each set-up tested with RSD% values ranging from 0.60 for the single C18 column to 0.58 and 1.09 for the two- and three-serially coupled columns, respectively (Table 1). The experimental pressure drop, to 70% IPA in ACN, was 20.5 MPa for the single C18 column to 59.5 MPa for the three serially coupled C18 columns. The set-up comprised of three serially coupled columns yielded backpressure values not compatible with a conventional HPLC instrument thus requiring the employment of ultra high pressure pumps. Being feasible, the addition of a fourth column was also tested but afterwards neglected compromising between resolution and retention times (data not shown). 3.2. Elucidation of the triacylglycerol composition in milk samples by (NARP)LC/APCI-MS Prior to the characterization of the triacylglycerol composition of the various milk and milk-derivative samples tested, GC–FID (gas chromatography–flame ionization detection) data were considered. For such a task, the results recently reported on the same goat, cow and human milk samples were used [44] whereas the fatty acid content of the buffalo mozzarella cheese sample is illustrated in Table S1. A complete list of FAs occurring in the identified TAGs investigated in this work is reported in Table 2. In all the samples investigated, the highest percentage of total FAs was represented by saturated FAs, followed by monounsaturated FAs and afterwards by polyunsaturated FAs. In particular the goat and cow milk samples contained the highest percentage of saturated FAs (roughly 74%), while the human milk sample only 46% of saturated FAs, and a nearly equal content of the monounsaturated ones (43%). The buffalo mozzarella cheese extract was similar to the goat and
cow milk samples with a percentage of saturated FAs roughly 70% followed by monounsaturated FAs. The relative abundance (%) of the protonated [M+H]+ ions can be very low (
