Embed Size (px)
Citation preview
Journal of Food Composition and Analysis 34 (2014) 68–74
Original Research Article
A rapid profiling of hypolipidemic agents in dietary supplementsby direct injection tandem mass spectrometry
Miranda Sertic, Ana Mornar *, Biljana Nigovic
University of Zagreb, Faculty of Pharmacy and Biochemistry, A. Kovacica 1, 10000 Zagreb, Croatia
A R T I C L E I N F O
Article history:
Received 30 September 2013
Received in revised form 5 March 2014
Accepted 5 March 2014
Keywords:
Natural cholesterol-lowering products
Dietary supplements safety
Regulatory challenge
Supplement composition
Direct injection mass spectrometry
Fingerprint analysis
Fragment analysis
Hyperlipidemia
Food composition
Food analysis
Food safety
A B S T R A C T
A new, fast-screening method for identification of various pharmacologically active ingredients in
cholesterol-lowering dietary supplements using direct injection mass spectrometry was developed. The
optimized method was proven to be useful for multitarget screening: 17 compounds of interest were
identified, including monacolin K and its acid form, cynarin, green tea catechines, genistein, daidzein,
guggulsterones, allicin and L-arginine. The method was successfully applied for the fingerprint analysis
of dietary supplements containing only one or several cholesterol-lowering agents including lovastatin
registered as a drug. According to the obtained results, it is apparent that cholesterol-lowering dietary
supplements are poorly standardized, as two samples did not contain active ingredients (monacolin K,
monacolin K acid form and cynarin). Moreover, the application of the proposed direct injection method
to quality control of cholesterol-lowering dietary supplements may be proposed for the routine analysis
of a large number of samples in the laboratories of regulatory agencies.
� 2014 Elsevier Inc. All rights reserved.
Contents lists available at ScienceDirect
Journal of Food Composition and Analysis
jo u rn al ho m epag e: ww w.els evier . c om / lo cat e/ j fc a
1. Introduction
Hyperlipidemia is a heterogeneous group of disorders com-monly characterized by an increased flux of free fatty acids, raisedlevels of triglycerides, low-density lipoprotein (LDL) cholesteroland apolipoprotein B, as well as by reduced plasma high-densitylipoprotein (HDL) cholesterol concentration. There are severalclasses of hypolipidemic drugs (statins, fibrates, nicotinic acid andits derivatives) which may differ in both their impact on thecholesterol profile and adverse effects (Mahamuni et al., 2012).Natural cholesterol-lowering agents are becoming popular as apossible alternative therapy for lowering plasma levels of totalcholesterol, especially for patients whose blood cholesterol level ismarginally high, but not high enough to warrant the prescription ofcholesterol-lowering medication.
Red mold rice is the fermented product of rice on which redmold (Monascus purpureus) has been grown. During the fermenta-tion process, 14 monacolins – compounds that inhibit 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in hepatic cholesterol synthesis – are produced.
* Corresponding author. Tel.: +385 1 4818 288; fax: +385 1 4856 201.
E-mail address: [email protected] (A. Mornar).
http://dx.doi.org/10.1016/j.jfca.2014.03.001
0889-1575/� 2014 Elsevier Inc. All rights reserved.
The most abundant monacolin is monacolin K, also known aslovastatin, the first commercially available statin drug fortreatment of hyperlipidemias (Fig. 1).
Scientists and health practitioners worldwide are in factquestioning whether red mold rice is a dietary supplement or adrug (Gordon and Becker, 2011). In recent years, artichoke (Cynara
scolymus, Asteraceae) has been introduced as a new natural lipid-lowering agent because cynarin (1,5-dicaffeoylquinic acid), themain active ingredient, shows the inhibition effect on HMG-CoAreductase enzyme as well as on phosphatidate phosphohydrolase(PAP) (Heidarian et al., 2011). One of the underlying mechanismsby which green tea (Camellia sinensis, Theaceae) catechines ((+)-catechin, (�)-epicatechin, (+)-catechin gallate, (�)-epicatechingallate, (+)-gallocatechin, (�)-epigallocatechin, (+)-gallocatechingallate, (�)-epigallocatechin gallate) reduce plasma cholesterollevel is up-regulation of LDL receptor activity (Spacil et al., 2010).Furthermore, it has been discovered that catechins stimulatecholesterol-7a-hydroxylase (CYP7A1) enzyme in human hepato-ma cells at mRNA level (Lee et al., 2008). The hypocholesterolemiceffect of soybean (Glycine max, Fabaceae) has been attributed to theproteins, fiber and phytochemicals including the soy isoflavonoids.Isoflavones, genistein and daidzein, have been shown to inhibitintestinal acyl coenzyme A cholesterol acyltransferaze (ACAT)activity in hepatocyte (Borradaile et al., 2002).
HO
O
O
O
H
HO
HO
O
OH
O
H
HO
OH
21
O
OH
O
OH
O
O
HO
O
HO
HO
OHHO
3
HO
OH
O
OH
OH
HO
OH
O
OH
4 5
OH
HO
OH
O
OH
OH
OH
6
OH
HO
OH
O
OH
OH
7
OH
OH
HO
OH
O
OH
O
OH
OH OH
O
OH
OH
OH
8
HO
OH
O
OH
O
OH
O
OH
OH
OH
9
HO
OH
O
OH
O
OH
O
OH
OH
OH
10
HO
OH
O
OH
O
OH
O
OH
OH
OH
11
OH OH
O
OH
O
HO
OH
12
O
OH
O
HO
13
O
O
H
O
O
H
14 15
S S
O
16
H2N NH
OH
NH
NH2
O
17
Fig. 1. Chemical structures of investigated natural cholesterol-lowering agents: Monacolin K (1), monacolin K acid form (2), cynarin (3), (+)-catechin (4), (�)-epicatechin (5),
(+)-catechin gallate (6), (�)-epicatechin gallate (7), (+)-gallocatechin (8), (�)-epigallocatechin (9), (+)-gallocatechin gallate (10), (�)-epigallocatechin gallate (11), genistein
(12), daidzein (13), E-guggulsteron (14), Z-guggulsteron (15), allicin (16) and L-arginine (17).
M. Sertic et al. / Journal of Food Composition and Analysis 34 (2014) 68–74 69
A number of studies have shown that two steroidal isomersknown as E- and Z-guggulsterone are active ingredients ofguggulipid, responsible for cholesterol-lowering effects of theherbal extract from the resin of the thorny plant Cammiphora
wighitii, Burseraceae (Ulbricht et al., 2005). Both isomers of
guggulsterone possess similar hypolipemic activity. Guggulster-ones selectively modulate farnesoid X receptor (FXR) geneexpression and positively regulate the expression of the cyto-chrome P450 7A1 and 8B1, thus inducing the cholesterolcatabolism into bile acids (Sinal and Gonzalez, 2002). Several
M. Sertic et al. / Journal of Food Composition and Analysis 34 (2014) 68–7470
studies have shown that the major active ingredient of garlic(Allium sativum, Amaryllidaceae) is allicin (2-propenyl-2-prope-nethiosulfinate) which inhibits the HMG-CoA reductase (Gebhardt,1993). More recently, L-arginine has been found in dietarysupplements in combination with other natural lipid-loweringagents, although there is not enough scientific evidence to supportits use for treatment of the patients with hyperlipidemia (Schulzeet al., 2009).
Many over-the-counter dietary supplements that promotecholesterol-lowering benefits are available on the market (Nijjaret al., 2010). These products are widely promoted, often withunsubstantiated claims of benefits and rarely with any mention ofpotential toxicity, interactions with drugs or quality control(Petroczi et al., 2011). Several studies have found markedvariability in the composition and content of active and/orcharacteristic compounds in these products, presumably verydifferent cholesterol-lowering efficacy and potential side effects(Gordon et al., 2010; Mornar et al., 2013; Nigovic et al., 2013).Taking into consideration tremendous expansion in their use anddramatic variation from labeled values found in several differentproducts, it is obvious that the quality control of dietarysupplements has become an important concern for both healthauthorities as well as the public (Au and Murphy, 2006).
Several methods for the determination of active and/orcharacteristic ingredients in cholesterol-lowering dietary supple-ments were have already been developed (Gouveia and Castilho,2012; Mornar et al., 2013; Nigovic et al., 2013). The mostcommonly used technique is liquid chromatography coupled toa diode array detector and/or a mass spectrometric detector (LC/DAD/MS). This technique has become a powerful tool to identifyeven trace amounts of active ingredients in complex matrices. Ithas proved to be an effective and convenient approach for thequalitative evaluation of these kinds of products, and could be usedfor the quality control of the target material as it yields detailedchemical information involving both the characteristic compoundand related constituents.
However, it is doubtful that the LC/DAD/MS technique presentsthe simplest and fastest procedure for fingerprint analysis. Ascounterfeiting of dietary supplements has become an importanthealth and economic problem, simple, fast and reliable analyticaltools for identification of characteristic compounds in dietarysupplements are highly desirable. In the past decade, we havewitnessed rapid development of direct-injection mass spectro-metric (MS) technology. The major advantage of the technique isthat the expensive, time-consuming and environmentally
Table 1List of investigated dietary supplements.
Product name Manufacturer Description
Antisklerin Dietpharm, Rakitje,
Croatia
Dietary supplement containing
lecithin (100 mg) and vitamin E
Artichoke Encian, Donji Stupnik,
Croatia
Artichoke leaf extract containin
Cholesterol
maintenance
KAL, Park City, USA Dietary supplement containing
Cynara scolymus leaf extract, L-
black pepper extract, ginger roo
cayenne extract
Green Tea ESI, Albissola Marina,
Italy
Dietary supplement containing
of polyphenols (40%)
No-colest
Omegasol
Specchiasol, Bussolengo,
Italy
Dietary supplement with decla
to 1.5% monacolins), citrus berg
Normolip 5 ESI, Albissola Marina,
Italy
Dietary supplement containing
monacolins, 150 mg gamma ory
Omelip Aktival, Ludbreg, Croatia Dietary supplement containing
1.5% of monacolin K, 1000 mg o
Red rice Encian, Donji Stupnik,
Croatia
Red mold rice containing at lea
Rizolip Aktival, Ludbreg, Croatia Red mold rice containing 1% of
unfriendly chromatographic separation is avoided and conse-quently the time of the analysis is significantly decreased.
To the best of our knowledge, a method for simultaneousidentification of several different cholesterol-lowering agents indietary supplements has not yet been proposed. Therefore, the aimof our work was to develop a new, fast screening method foridentification of various active ingredients in cholesterol-loweringdietary supplements using direct injection electrospray ionization-mass spectrometry (ESI-MS).
2. Materials and methods
2.1. Chemicals and reagents
Methanol, HPLC grade, was purchased from Merck (Darmstadt,Germany). Ammonium hydroxide solution and formic acid,reagent grade, were purchased from Sigma–Aldrich (St. Louis,MO, USA). The ultrapure water was prepared with a Mili-Q waterpurification system (Milipore, Bedford, MA, USA).
2.2. Sample preparation
Nine dietary supplements containing natural cholesterol-lowering agents were supplied from a local community pharmacy.The dietary supplements analyzed in this work were in multipledosage forms including: tablets, capsules, liquid capsules andsoftgels. A list of the products together with the manufacturer andcontent description is given in Table 1.
Ten tablets were finely ground and used in further investiga-tions. Likewise, the content of 10 capsules or softgels was pooledand homogenized. A sample of each weighing 1 g was transferredto a 15 mL tampon centrifuge tube. The suspensions wereextracted with 10.0 mL of 80% methanol for 60 min at roomtemperature using ultrasonic bath. Finally, the solution wascleared by centrifugation at 3000 � g for 10 min at 25 8C, thesupernatant was collected and then filtered through a 0.45 mmChromafil membrane filter (Macherey-Nagel, Duren, Germany)before injection. Three different batches of each sample wereinvestigated and each sample was prepared in triplicate.
2.3. Mass spectrometry
The analysis of the samples was performed by infusing samplesolutions in 80% methanol at low rate of 5 mL/min, via an externalsyringe pump (KD Scientific Inc., Holliston, USA) directly connected
Sample type
350 mg of garlic oil standardized content of allicin (0.1%),
(51 mg)
Liquid
capsule
g more than 5% of cynarin Tablet
0.6 g of red mold rice, Commiphora mukul gum extract,
arginine, Non-GMO soy bean concentrate, gamma oryzanol,
t extract, rosemary leaf extract, turmeric root extract and
Tablet
a dried extract of green tea standardized content Capsule
red amount of 200 mg of red fermented rice (standardized
amia, fatty acids, GMO free soy lecitin and chlorofil
Softgel
100 mg of red fermented rice standardized to 3% of
zanol, 120 mg phytosterols and 5 mg policozanol
Capsule
200 mg of dried red mold rice extract containing
f fish oil (18% EPA, 12% DHA), vitamin C and vitamin E
Liquid
capsule
st 1.5% of monacolin K Capsule
monacolin K Capsule
Table 2The fragment ions and MRM transitions used for the identification of active ingredients in cholesterol-lowering dietary supplements.
Analyte MRM m/z transition Fragment ions
Monacolin K 405 ! 285 303, 285, 243, 225, 199
Monacolin K acid form 423 ! 285 405, 321, 303, 285, 267, 243, 225, 199
Cynarin 515 ! 353 353, 191
Catechin/epicatechin 291 ! 123 273, 139, 123
Catechin gallate/epiactechin gallate 443 ! 123 291, 273, 139, 123
Gallocatechin/epigallocatechin 307 ! 139 289, 139
Gallocatechin gallate/epigallocatechin gallate 459 ! 139 307, 289, 139
Genistein 271 ! 253 253, 243, 225, 215, 153, 119
Daidzein 255 ! 237 237, 227, 209, 199, 137, 103
E-guggulsterone/Z-guggulsterone 313 ! 295 295, 277, 249
Allicin 163 ! 121 121, 73
L-arginine 175 ! 70 158, 116, 70
M. Sertic et al. / Journal of Food Composition and Analysis 34 (2014) 68–74 71
to the mass spectrometer. All experiments were performed on an LC/MSD ion trap (Agilent Technologies, Waldbronn, Germany) massspectrometer equipped with an electrospray ionization (ESI) sourceand an ion trap analyzer system controlled by LC/MSD Trap (AgilentTechnologies, Waldbronn, Germany) software. The MS system wascalibrated using the Tune mix (Agilent Technologies, Waldbronn,Germany). The ESI source was operated in positive- and negative-ionization mode. The source dependent parameters were as follows:capillary voltage 3.5 kV; source temperature 325 8C; nebulizer gas,nitrogen, at 15 psi and 5 L/h. Helium was used for fragmentation andthe MSn studies were carried out by keeping the collision energy at30%. Continuous mass spectra were obtained by scanning from 10 to1100 m/z. The 10,000 ions were trapped in analyzer and theaccumulation time was set at 200 ms. Analysis of the samples wasperformed in multiple reaction monitoring mode (MRM), using theprecursor ion and the corresponding product ions (Table 2).
3. Results and discussion
3.1. Method development
The operating MS parameters of the ion source were optimizedto obtain the best performance of mass spectrometer for theidentification of all active ingredients. Parameters such as capillaryvoltage, nebulizer gas pressure, dry gas flow and dry temperaturewere optimized. The optimization of MS parameters wasperformed using mixture of samples containing all investigatedcholesterol-lowering compounds. The above described extractionprocedure of analytes was chosen on the basis of previouslypublished methods (Mornar et al., 2013; Nigovic et al., 2013).
To obtain the optimum capillary voltage, a voltage range from2.5 to 4.0 kV was examined at increments of 0.5 kV. The greatestsignal intensity for all analytes was obtained when the voltage wasset at 3.5 kV. An optimum value for the flow and temperature ofdrying gas was carefully selected to obtain the highest possiblesensitivity. First, the optimization procedure for drying gastemperature was carried out by modifying the temperature from250 to 350 8C at increments of 25 8C. The greatest signal intensityfor all analytes was obtained when temperature of the drying gaswas set at 325 and 350 8C. The temperature of 325 8C was chosenfor further investigations in order to avoid the decomposition ofanalytes at high temperatures. At this fixed temperature setting,the drying gas flow was varied from 1 to 10 L/min. The results fromthese experiments showed the greatest signal intensity at flow rateof 5 L/min. The nebulizer pressure was increased from 10 to 60 psiat increments of 5 psi. Finally, the nebulizer pressure was set at15 psi as a fine even mist without fluctuation at the tip of the sprayneedle, as the best sensitivity for all analytes. The pH of thesamples was adjusted so that the acidic and basic analytes wereionized. The influence of various buffers, acid and base additives onthe ionization process of analytes was investigated. The obtained
preliminary results have shown the highest signal intensity of allanalytes using 0.1% formic acid or 0.1% ammonium hydroxidesolution.
As the ion trap is a storage device, it is possible to accumulateweak signals over an extended period of time. It was expected thatthe investigated dietary supplements contain diverse compositionand content of cholesterol-lowering agents; therefore, theaccumulation time was increased from 10 ms to 1 s. For most ofthe analytes the greatest signal intensity was obtained using theaccumulation time of 200 ms. The number of ions trapped in theanalyzer varied from 10,000 to 50,000. As the number of ionsincreased, reduced mass resolution and accuracy was obtained;thus the number of ions in the trap was maintained at 10,000.
3.2. Fingerprinting of active constituents by ESI-MS/MS technique
To select the most abundant parent and fragment ions ascandidates for identification by MRM analysis of samples, thefragmentation pattern of each analyte was investigated. The massspectrometric behavior of all analytes was studied using positiveand negative ionization mode.
Although in red fermented rice 13 other monacolins could befound, we found in our previous study that the main activecomponents which contribute to up to 99.8% of the total quantityof monacolins are monacolin K and its acid form (Mornar et al.,2013). Therefore, the study was designed to investigate thepresence of these two monacolins in commercially availabledietary supplements. Monacolin K produced the most abundantion corresponding to the protonated molecule (M+H)+ at m/z 405,which afterwards generated a complex ESI-MS/MS spectrum withthe formation of multiple fragment ions at m/z 303, 285, 243, 225and 199. Fragmentation of the protonated molecule generated anion at m/z 303 formed after the loss of the ester side-chain and theion at m/z 285 obtained by the neutral loss of a water moleculefrom the ion at m/z 303 (Fig. 2A). The acid form of monacolin Kclearly showed the characteristic protonated molecule (M+H)+ atm/z 423. The ESI-MS/MS spectrum of ion at m/z 423 showed thefragment ions at m/z 405, 321, 303, 285, 267, 243, 225 and 199.According to these results, the major fragments were obtained bythe loss of a water molecule and/or the ester side-chain (Fig. 2B)(Mornar et al., 2013).
Cynarin, the major hypolipidemic agent in artichoke leafextract, gave the deprotonated molecule (M�H)�(m/z 515). Toconfirm the identity of cynarin, fragmentation of deprotonatedmolecule was performed and the obtained ESI-MS/MS spectrumshowed the presence of the two major fragment ions at m/z 353and 191. These results suggest that the major fragments wereobtained by the loss of one or two caffeic units, respectively(Fig. 3).
Significantly better sensitivity of the method for the maincholesterol-lowering components of green tea was obtained using
Fig. 2. ESI-MS/MS spectra and proposed fragmentation patterns of monacolin K (A) and monacolin K acid (B). The optimized conditions are described in Section 2.3.
M. Sertic et al. / Journal of Food Composition and Analysis 34 (2014) 68–7472
positive ion detection mode. In positive ion detection modecatechin and epicatechin gave protonated molecule at m/z 291. TheESI-MS/MS spectrum gave the base peak at m/z 123 and otherprominent fragments at m/z 273 and 139. Catechin gallate andepicatechin gallate gave the same protonated molecule at m/z 443.After the cleavage of gallate and formation of the fragment at m/z291, the fragmentation pattern of catechin gallate and epicatechingallate were similar to the fragmentation of catechin andepicatechin with prominent fragments seen at m/z 273, 139 and123. The difference between catechin/epicatechin and gallocate-chin/epigallocatechin is simply an additional hydroxyl group onthe phenol ring B; therefore, as was expected, the fragmentationpatterns were similar. Gallocatechin gallate and epigallocatechingallate gave the same protonated molecule at m/z 459. The ESI-MS/MS spectrum of the protonated molecule gave the base peak at m/z139 and other prominent fragments at m/z 307 and 289. Thefragment at m/z 307 corresponds to the loss of gallate attached toring C while the fragment at m/z 289 is obtained by the loss of awater molecule from the fragment at m/z 307. Similarly togallocatechin and epigallocatechin, the major fragment at m/z 139
Fig. 3. ESI-MS/MS spectrum and proposed fragmentation pattern of
was obtained by fragmentation of the fused ring system (Spacilet al., 2010).
The positive ionization mode showed 2–3 times bettersensitivity than the negative ionization mode for genistein anddaidzein, the most abundant isoflavonoids in soybean. In positiveionization mode, genistein was characterized by the cation ioncorresponding to protonated molecule at m/z 271. Fragmentationvia a retro-Diels–Alder (RDA) reaction gave product ions at both m/z 153 and 119. The ESI-MS/MS spectrum of genistein also showed anumber of other fragments (m/z = 253, 243, 225, 215) obtained bythe losses of water and/or carbonyl group which could occur frommultiple sites of the molecule. The mass fragmentation pattern ofdaidzein was similar to the fragmentation of genistein. Theprotonated molecule at m/z 255 generated a complex ESI-MS/MSspectrum with the formation of multiple fragment ions. Thefragments at m/z 137 and 103 were obtained via an RDA reactionwhile the fragments at m/z 237, 227, 209 and 199 were obtained bythe losses of water and/or carbonyl group.
Since active ingredients of guggulipid, E- and Z-guggulsterone,have an equal molecular mass, both analytes gave the same
cynarine. The optimized conditions are described in Section 2.3.
Fig. 4. Fingerprint of sample containing several natural cholesterol-lowering herbs. The optimized conditions are described in Section 2.3.
Table 3List of ions found in investigated dietary supplements.
Sample no. MRM transitions (ion intensity � RSD %)a
1 163 ! 121 (2.2 � 103� 5.3%)
2 515 ! 353 (1.2 � 104� 1.4%)
3 405 ! 285 (1.9 � 103� 3.7%)
423 ! 285 (2.1 � 103� 2.5%)
313 ! 295 (8.1 � 102� 6.5%)
175 ! 70 (8.3 � 102� 8.3%)
271 ! 253 (8.4 � 102� 8.9%)
255 ! 237 (8.5 � 102� 9.1%)
515 ! 353 (expected but not detected)
4 291 ! 123 (8.3 � 103� 2.3%)
443 ! 123 (7.3 � 103� 2.2%)
307 ! 139 (2.3 � 103� 4.5%)
459 ! 139 (5.3 � 103� 3.7%)
5 405 ! 285 (1.1 � 103� 4.7%)
423 ! 285 (1.5 � 103� 4.0%)
6 405 ! 285 (4.5 � 103� 2.7%)
423 ! 285 (4.4 � 103� 4.1%)
7 405 ! 285 (expected but not detected)
423 ! 285 (expected but not detected)
8 405 ! 285 (8.5 � 102� 6.8%)
423 ! 285 (8.4 � 102� 6.1%)
9 405 ! 285 (7.4 � 102� 5.2%)
423 ! 285 (7.1 � 102� 6.3%)
a Relative standard deviation (n = 3).
M. Sertic et al. / Journal of Food Composition and Analysis 34 (2014) 68–74 73
protonated molecule (M+H)+ at m/z 313. This protonated moleculegenerated a simple ESI-MS/MS spectrum with the formation of thefragment ion at m/z 295, obtained by loss of a water molecule.Furthermore, two other fragments were observed at m/z 277 and249.
Allicin showed the protonated molecule (M+H)+ at m/z 163. TheESI-MS/MS spectrum of the obtained protonated molecule showedthe presence of the two major fragment ions at m/z 121 and 73.These two fragment ions can result from the dissociation of theallicin radical cation. Ion at m/z 121 is assumed to be formed due tothe loss of propylene from protonated allicin, while ion at m/z 73 isassigned to the allyl sulfide cation (Zhang, 2012).
L-arginine gave the protonated molecule (M+H)+ at m/z 175.This protonated molecule generated a simple ESI-MS/MS spectrumwith the formation of a base fragment ion at m/z 70 and severalminor fragments at m/z 158 and 116. It is assumed that the mostintense product ion most likely corresponds to a pyrrolinium ion asa cyclization product (Shin et al., 2011).
3.3. Analysis of natural cholesterol-lowering agents in dietary
supplements
The usefulness of the proposed method was evaluated byanalysis of nine dietary supplements commercially available incommunity pharmacies and local health food stores. Five of theinvestigated dietary supplements consisted of only one naturalcholesterol-lowering agent, while four of the samples had quitecomplex matrices, consisting of several cholesterol-loweringagents. First, all of the samples were screened in order to obtainthe characteristic full-scan mass spectrum of each dietarysupplement. The obtained mass spectra represented the sum ofthe intensities of all ions detected during the data acquisitionperiod. The full-scan mass spectrum of dietary supplementcontaining a mixture of natural cholesterol-lowering agents ispresented in Fig. 4. The ESI source was operated in positiveionization mode. Several ions corresponding to protonatedmolecules can be identified (m/z = 423 – monacolin K acid, m/z = 405 – monacolin K, m/z = 313 – E- or Z-guggulsterone and m/z = 175 – L-arginine). Afterward, the samples were investigatedusing MRM operation mode which works like a double mass filter,resulting in unmatched selectivity and sensitivity. Using MRMmode very little noise or matrix interference was detected. Theproposed fast screening method was proven to be useful formultitarget screening as 17 compounds of interest could beidentified in a single run. In order to evaluate the reproducibility ofour method, 20 injections of all samples were carried out. Theintensities of all ions did not decrease by more than 1%. Themethod was successfully applied for dietary supplementscontaining either one or several natural cholesterol-loweringagents (Table 3).
4. Conclusion
Regardless of the high complexity of the analyzed samples, thissimple and inexpensive sample preparation procedure wasemployed. Finally, the great advantage of the proposed directinjection method is that an expensive, time-consuming andenvironmentally unfriendly chromatographic separation wasunnecessary. The major limitation of our study is that theproposed method, although fast, inexpensive and simple, presentsonly a qualitative method. In order to determine concentrations ofanalytes in the samples, LC/MS/MS technique should be used.
Our results showed that in one sample, containing artichokeleaf extract, the major active ingredient cynarin was not found.Furthermore, in another investigated dietary supplement, contain-ing red fermented rice, none of the monacolins were found.According to these results it may be deduced that cholesterol-lowering dietary supplements are poorly standardized, resulting ininconsistent composition and pharmacological activity.
The use of natural cholesterol-lowering agents has expanded inrecent years and is expected to increase even further in the yearsahead because hyperlipidemias and cardiovascular diseases arebeing diagnosed and treated more frequently. Therefore, thequality control of these products is of great importance. This typeof fast screening method could be very useful for manufacturers tocontrol the quality of their purchased raw materials prior to its
M. Sertic et al. / Journal of Food Composition and Analysis 34 (2014) 68–7474
formulation and tableting or encapsulating and their finalproducts, as well as for regulatory agencies due to its simplicityand applicability for multitarget screening of some of the mostfrequently used natural cholesterol-lowering agents.
Acknowledgements
This work was supported through a grant (Investigation of newmethods in analysis of drugs and bioactive substances) from theMinistry of Science, Education and Sports of the Republic ofCroatia.
References
Au, D.L.M.T., Murphy, S.P., 2006. Creating a single combined composition table forfoods and dietary supplements. Journal of Food Composition and Analysis 19,S81–S85.
Borradaile, N., De Dreu, L., Wilcox, L., Edwards, J., Huff, M., 2002. Soya phytoestro-gens, genistein and daidzein, decrease apolipoprotein B secretion from HepG2cells through multiple mechanisms. Biochemical Journal 366, 531–539.
Gebhardt, R., 1993. Multiple inibitory effects of garlic extracts on cholesterolbiosynthesis in hepatocites. Lipids 28, 613–619.
Gordon, R.Y., Becker, D.J., 2011. The role of red yeast rice for the physician. CurrentAtherosclerosis Report 13, 73–80.
Gordon, R.Y., Cooperman, T., Obermeyer, W., Becker, D.J., 2010. Marked variability ofmonacolin levels in commercial red yeast rice products. Archives of InternalMedicine 170, 1722–1727.
Gouveia, S.C., Castilho, P.C., 2012. Phenolic composition and antioxidant capacity ofcultivated artichoke, Madeira cardoon and artichoke-based dietary supple-ments. Food Research International 48, 712–724.
Heidarian, E., Jafari-Dehkordi, E., Seidkhani-Nahal, A., 2011. Lipid-lowering effect ofartichoke on liver phosphatidate phosphohydrolase and plasma lipids in hyper-lipidemic rats. Journal of Medicinal Plants Research 5, 4918–4924.
Lee, M.S., Park, J.Y., Freake, H., Kwun, I.S., Kim, Y., 2008. Green tea catechin enhancescholesterol 7 alpha-hydroxylase gene expression in HepG2 cells. British Journalof Nutrition 99, 1182–1185.
Mahamuni, S.P., Khose, R.D., Menaa, F., Badole, S.L., 2012. Therapeutic approaches todrug targets in hyperlipidemia. Biomedicine 2, 137–146.
Mornar, A., Sertic, M., Nigovic, B., 2013. Development of a rapid LC/DAD/FLD/MS(n)
method for the simultaneous determination of monacolins and citrinin in redfermented rice products. Journal of Agricultural and Food Chemistry 61, 1072–1080.
Nigovic, B., Sertic, M., Mornar, A., 2013. Simultaneous determination of lovastatinand citrinin in red yeast rice supplements by micellar electrokinetic capillarychromatography. Food Chemistry 138, 531–538.
Nijjar, P.S., Burke, F.M., Bloesch, A., Rader, D.J., 2010. Role of dietary supplements inlowering low-density lipoprotein cholesterol: a review. Journal of ClinicalLipidology 4, 248–258.
Petroczi, A., Taylor, G., Naughton, D.P., 2011. Mission impossible? Regulatory andenforcement issues to ensure safety of dietary supplements. Food and ChemicalToxicology 49, 393–402.
Schulze, F., Glos, S., Petruschka, D., Altenburg, C., Maas, R., Benndorf, R., Schwed-helm, E., Beil, U., Boger, R.H., 2009. L-Arginine enhances the triglyceride-lower-ing effect of simvastatin in patients with elevated plasma triglycerides.Nutrition Research 29, 291–297.
Shin, S., Fung, S.-M., Mohan, S., Fung, H.-L., 2011. Simultaneous bioanalysis of L-arginine, L-citrulline, and dimethylarginines by LC–MS/MS. Journal of Chroma-tography B 879, 467–474.
Sinal, C.J., Gonzalez, F.J., 2002. Guggulsterone: an old approach to a new problem.Trends in Endocrinology and Metabolism 13, 275–276.
Spacil, Z., Novakova, L., Solich, P., 2010. Comparison of positive and negative iondetection of tea catechins using tandem mass spectrometry and ultra highperformance liquid chromatography. Food Chemistry 123, 535–541.
Ulbricht, C., Basch, E., Szapary, P., Hammerness, P., Axentsev, S., Boon, H., Kroll, D.,Garraway, L., Vora, M., Woods, J., 2005. Guggul for hyperlipidemia: a review bythe natural standard research collaboration. Complementary Therapies inMedicine 13, 279–290.
Zhang, X., 2012. Mass spectrometric and theoretical studies on dissociation of the S–S bond in the allicin: homolytic cleavage vs. heterolytic cleavage. Journal ofMolecular Structure 1020, 63–69.
