Research Article

Received: 9 October 2012 Revised: 9 January 2013 Accepted: 13 January 2013 Published online in Wiley Online Library

Rapid Commun. Mass Spectrom. 2013, 27, 773–783

Structural characterization of biocompatible lipoic acid–oligo-(3-hydroxybutyrate) conjugates by electrospray ionization massspectrometry

Magdalena Maksymiak1, Renata Debowska2, Katarzyna Jelonek1, Marek Kowalczuk1 andGrazyna Adamus1*1Polish Academy of Sciences, Centre of Polymer and Carbon Materials, 34 M. Curie-Sklodowskiej St., 41-819 Zabrze, Poland2Dr Irena Eris Centre for Science and Research, 107A Pulawska St., 02-595 Warszawa, Poland

RATIONALE: Currently, most of the antioxidants and free radical neutralizers used in cosmetic compositions areabsorbed quickly into deeper layers of skin, and then carried away by the blood stream. It would be beneficial to delaythe penetration of antioxidants to the deeper layers of skin to control their delivery and release.METHODS: Recently, growing attention has been paid to the attachment of cosmetics to specific polymer carriers.Biodegradable and biocompatible conjugates of oligo-3-hydroxybutyrate with lipoic acid were obtained via the anionicring-opening oligomerization of (R,S)-b-butyrolactone initiated by lipoic acid potassium salt. The structure of theresulting conjugates as well as their water-soluble hydrolytic degradation products were established at the molecularlevel by electrospray ionization mass spectrometry (ESI-MSn) supported by 1H NMR analyses.RESULTS: The structural studies, performed with the aid of ESI-MSn, confirmed that the lipoic acid was covalentlybound to oligo-3-hydroxybutyrate chains through hydrolyzable ester bonds. Furthermore, hydrolytic degradationstudies of the bioconjugates provided detailed insight into the hydrolysis process, allowing the identification of thedegradation products and confirming the release of a-lipoic acid. Cytotoxicity tests demonstrated that the conjugateswere non-toxic.CONCLUSIONS: Detailed molecular structural studies of new polymeric delivery systems of lipoic acid wereperformed by ESI-MS. ESI-MS proved to be an excellent technique for the evaluation of hydrolytic degradation productsof the conjugates and for monitoring the release of lipoic acid. The results obtained contribute significantly to thecharacterization of biocompatible LA-OHB conjugates with potential applications in cosmetology. Copyright © 2013John Wiley & Sons, Ltd.

(wileyonlinelibrary.com) DOI: 10.1002/rcm.6509

a-Lipoic acid ((R)-5-(1,2-dithiolan-3-yl)pentanoic acid) is auniversal antioxidant, meaning that it captures naturallyoccurring but harmful chemicals known as free radicals.Lipoic acid (LA) shows antioxidant activity in both, waterand fatty tissues, giving it an unusually broad spectrum ofantioxidant action.[1–3] By virtue of its numerous advanta-geous properties, including good water- and fat-solubility aswell as moisturizing, anti-inflammatory, photoprotective,and antioxidant properties, LA is commonly used in cos-metics to produce a wide range of facial and body care pro-ducts as well as skin protection formulas with anti-wrinkleeffects.[4]

The development of new cosmetic delivery systems has beenmost heavily influenced by two factors: the development ofnew synthetic surfactants, and the understanding of the struc-ture of the skin and its function in relation to percutaneous

* Correspondence to: G. Adamus, Polish Academy of Sciences,Centre of Polymer and Carbon Materials, 34 M. Curie-Sklodowskiej St., 41-819 Zabrze, Poland.E-mail: grazyna.adamus@cmpw-pan.edu.pl; gadamus@cmpw-pan.edu.pl

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absorption, both of which occurred about 50 years ago. Amongthe most well-understood cosmetic delivery systems are:vesicular systems (liposomes, silicone vesciles), emulsions(microemulsions, multiple emulsions, nanoemulsions, picker-ing emulsions, gel emulsions), particulate systems (microparti-cles, nanoparticles, and matrices), and cyclodextrin complexes.Biodegradable polymers are particularly popular componentsin cosmetic products based on pickering emulsions andnanoparticles.[5–8]

Recently, the strategy of attaching cosmetics to specific poly-mer carriers in order to increase the duration of their activity viaslow release and specific targeting, especially in the skin layers,has attracted substantial attention.Most of the antioxidants andfree radical neutralizers used in cosmetics are absorbed quicklyinto deeper layers of the skin and are then carried away by thebloodstream. It would be preferable for antioxidants and freeradical neutralizers to be temporarily bound to specific carriersin order to retard their penetration into deeper layers of the skinand therefore promote intracellular antioxidant and free radicalneutralizing activity.[9]

There are few examples in the literature describing the useof poly(a-hydroxy acid) as a polymer carrier for the deliveryof biologically active compounds within the skin.[10]

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Scheme 1. Synthesis of oligomers obtained via the ring-openingoligomerization of b-butyrolactone in the presence of lipoicacid potassium salt.

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However, cosmetic delivery systems based on biodegradablepolymers, especially poly(b-hydroxy acids), have not yetbeen fully investigated.It has been found that low molecular weight, tailor-made

poly-3-hydroxybutyrate (PHB) can be synthesized via theanionic ring-opening polymerization of b-butyrolactoneinitiated by sodium or potassium carboxylates activated withcrown ethers or highly polar solvents.[11,12] Synthetically pre-pared oligo(3-hydroxybutyrate)s (OHBs) have been found tobe non-toxic[13] and have been used as carriers covalentlybound to suitable drug precursors. Detailed mass spectrome-try studies on the oligo(3-hydroxybutyrate) covalently boundto penicillin G, obtained via anionic ring-opening oligomeri-zation (ROO) of b-butyrolactone, have been reported.[14]

Using the same approach, aspirin- and ibuprofen-OHB conju-gates have been prepared recently, both of which were foundto be less toxic than the free drug compounds.[15,16]

The understanding of structure-property relationships isessential for the successful application of polymeric materialsin biomedical applications such as tissue engineering, con-trolled drug delivery, and cosmetic modifications. Powerfulcharacterization techniques are needed to obtain the requiredstructural information. In recent years, mass spectrometry hasbecome a routine analytical tool for the characterization ofpolymers, complementing nuclear magnetic resonance(NMR) and other traditional techniques by providing detailedstructural information about individual macromolecules.In the past decade, mass spectrometry techniques have

been applied to the molecular-level structural characterizationof homo- and copolyesters[17–22] as well as their degradationproducts.[23–25]

Electrospray ionization ion trap multistage mass spectro-metry (ESI-MSn) has been successfully applied to the analysisof individual aliphatic polyesters chains, in particularthose consisting of synthetic PHB analogues as well as theiroligomeric degradation products.[26–28] The application of ESI-MSn fragmentation analysis to the molecular-level characteriza-tion of individual chains of oligo([R,S]-3-hydroxybutyrate)conjugates with selected pesticides has been also recentlyreported by some of us.[29]

In the present study, we report the synthesis of conjugates ofoligo-3-hydroxybutyrate with lipoic acid via ring-opening oligo-merization of racemic b-butyrolactone initiated by lipoic acidpotassium salt. The structure of individual macromolecules ofthe resulting conjugates (including the chemical structure of theirend groups) was determined with the aid of an electrosprayionization tandem mass spectrometry technique (ESI-MSn) sup-ported by 1HNMR spectroscopy.Moreover, hydrolytic degrada-tion tests of selected bioconjugates were performed underlaboratory conditions. Through the monitoring of the hydrolyticdegradation process, identification of the degradation productsformed and the determination of their molecular structure usingESI-MSn were achieved. In addition, preliminary cytotoxicitytests were performed on the synthesized conjugates.

EXPERIMENTAL

Materials

(R,S)-b-Butyrolactone (98%, Aldrich) was purified as describedpreviously.[11,12] (�)-a-Lipoic acid (synthetic, ≥99% (titration),powder (Sigma)) and potassium hydroxide (pure pa, POCH

wileyonlinelibrary.com/journal/rcm Copyright © 2013 John Wi

SA) were used without additional purification. Ethanol (purepa, 99.8%, POCH SA) was used without additional drying.Dimethyl sulfoxide (DMSO, 99.8%, Aldrich) was dried overbarium oxide, fractionally distilled at 10 mmHg, and storedover 4 Å molecular sieves prior to use.

Syntheses of conjugates of a-lipoic acid with oligo-3-hydroxybutyrate (LA-OHB)

Synthesis of a-lipoic acid potassium salt (potassium a-lipoate)

a-Lipoic acid (0.03 mol) was dissolved in 30 mL of ethanoland combined with 300 mL of aqueous potassium hydroxide(0.1 mol/L). The mixture was heated and filtered, and the fil-trate was freeze-dried to yield 6.4 g of yellow amorphouspotassium a-lipoate.

Oligomerization of b-butyrolactone in the presence of a-lipoic acidpotassium salt

LA-OHB conjugates were synthesized via ROO of (R,S)-b-butyrolactone initiated by LA potassium salt. The reactionwas conducted in DMSO at 18 �C in a glass reactor that wasflamed and dry argon-purged prior to use. After 3 h of stir-ring, b-butyrolactone monomer was added to the reactor con-taining the required amount of initiator in DMSO. In eachexperiment, the monomer concentration was equal to1 mol/L. The average molecular mass of the conjugates wascontrolled by the monomer-initiator ratio. Progress of thepolymerization was monitored by Fourier transform infraredspectroscopy (FTIR) based on the intensity of the signals aris-ing from the carbonyl groups of the monomer and oligomerat 1815 and 1735 cm–1, respectively. When the reaction wascompleted, the calculated amount of HCl in DMSO wasadded to the reactor, allowed to react for 10 min, and the mix-ture was freeze-dried. The resulting product was re-dissolvedin CHCl3 and washed six times with distilled water in orderto remove potassium chloride and DMSO residue. Theobtained LA-OHB conjugates were precipitated in hexaneand dried under vacuum for 48 h. The synthesis of the oligo-mers obtained via ROO of b-butyrolactone in the presence ofLA potassium salt is outlined in Scheme 1.

MTT cytotoxicity assay

Keratinocytes (epidermoid carcinoma cell line KB, AmericanType Culture Collection) were grown in standard RPMI med-ium (Sigma-Aldrich) supplemented with 10% fetal calf serum(Gibco), 2 mM L-glutamine (Sigma-Aldrich), and antibiotics(100 U/mL penicillin, 0.25 mg/mL streptomycin sulfate,Sigma-Aldrich) at 37 �C in 5% CO2.

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ESI-MS structural studies on LA-OHB conjugates

The synthesized conjugates (Samples 1–4, Table 1) wereused to prepare stock solutions at concentrations of 10, 30,and 90 mg/mL in DMSO, and were added the into culturemedium immediately before use (final concentrations were1, 3, and 9 mg/mL, respectively). Control cells were treatedwith the same concentration of DMSO.For 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide (MTT) cytotoxicity assays, cells were seeded at adensity of 2000 cells/well in 96-well plates and incubatedfor 24 h to allow attachment. The medium was aspiratedand replaced with media containing increasing concentra-tions of LA-OHB conjugates. Each concentration was testedsix times, and experiments were repeated at least three times.After treatment, the medium was aspirated and the cells wereincubated for 3 h in RPMI with 0.5 mg/mL MTTat 37 �C in ahumidified atmosphere with 5% CO2. Then the medium wasaspirated, and 100 mL of 0.04 M HCl in isopropanol wasadded to each well in order to dissolve any formazan crystals.The absorbance of the samples was measured using a PowerWave XS Microplate Reader (Bio-tek Instruments Inc.) at570 nm.

Hydrolytic degradation tests of LA-OHB conjugates

Preliminary hydrolytic degradation tests in deionized water(pH 7) at 37 �C were performed for the selected LA-OHB con-jugates (Sample 2, Table 1). The water solubility of the biocon-jugates depended on molecular weight; low molecularweight LA-OHB conjugates (Mn,GPC = 900 Da) were fullysoluble in water. Approximately 25 mg of LA-OHB conjugateand 6 mL of water were combined in 20 mL glass vials, whichwere sealed with septa and placed in a thermostatically con-trolled incubator set to 37 �C. Aliquots were withdrawn intriplicate from the test environment after 3, 6, 24, 30, 48,and 168 h and analyzed by ESI-MS in negative ion mode.

Table 1. Results of the anionic oligomerization of (R,S)-b-butyrDMSO at 18 �Ca

Sample[I]0 10

–1

[mol/L] Mn,thb Mn,GPC

c M

1* 2.0 636 6502* 1.2 922 9003** 1.0 1066 11004** 0.5 1926 2100aInitial concentration of b-butyrolactone [C0] was 1.0 mol/L into 100%.

bMn,th is the theoretical molecular weight of conjugates, calcula

Mn;th ¼ C0½ �I0½ � � 86 þ M

where [C0] and [I0] are the initial concentrations of monomer anb-butyrolactone monomer and Ma-LA end group is the molar maequal to 205 and 1 is the molar mass of the proton.

cDetermined by gel-permeation chromatography (GPC) relativedCalculated from 1H NMR spectra.eEstimated on the basis of GPC measurements.*Samples fully soluble in water.**Samples soluble in methanol/water (1:1 v/v).

Copyright © 2013 JRapid Commun. Mass Spectrom. 2013, 27, 773–783

Measurements

Gel permeation chromatography (GPC) analysis

Number-average and weight-average molar mass and molarmass distribution (Mw/Mn) relative to polystyrene standardswere determined by GPC conducted in chloroform at 35 �Cwith a flow rate of 1 mL/min, using an isocratic pump (VE1122, Viscotek), a PLgel 5 mmMIXED-E (Polymer Laboratories)ultra-high efficiency column, and a Shodex SE 61 differentialrefractive index detector. The injection volume was 100 mL ofsample in chloroform (0.3%w/v). Polystyrene standards (Poly-mer Laboratories) with narrow molecular weight distributionswere used to generate a calibration curve.

NMR analysis1H NMR spectra were recorded using a Bruker-Avance600 MHz multinuclear magnetic resonance spectrometer. Theanalyses were run in CDCl3 using tetramethylsilane (TMS) asan internal standard.

FTIR analysis

FTIR spectra were recorded using a FTS 40A Bio-Rad spectro-meter at room temperature.

Electrospray mass spectrometry (ESI-MSn) analysis

Electrospray mass spectrometry analysis was performedusing a Finnigan LCQ ion trap mass spectrometer (Finnigan,San Jose, CA, USA). The conjugates (Samples 1–4, Table 1)were dissolved in either methanol/water (1:1 v/v) or chloro-form/methanol (1:1 v/v, samples with Mn >2000 g/mol).The water-soluble degradation products were analyzed inmethanol/water (1:1 v/v). The solutions were introducedinto the ESI source by continuous infusion at a rate of 10

olactone initiated by a-lipoic acid potassium salt (a-LAK) in

NMRd Mw/Mn

eReaction time

[h]Yield[%]

600 1.18 30 93800 1.13 72 921100 1.18 72 922200 1.14 120 94

each experiment; conversion in each experiment was equal

ted according to the following formula:

a-LA end group þ 1;

d the initiator, respectively; 86 g/mol is the molar mass of thess of the end groups originated from a-lipoic acid which is

to polystyrene standards with a low dispersity.

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mL/min using the instrument syringe pump. The LCQ ESIsource was operated at 4.5 kV and the capillary was heatedto 200 �C. Nitrogen was used as the nebulizing gas. For ESI-MS/MS experiments, monoisotopic ions of interest were iso-lated in the ion trap and collisionally activated using heliumdamping gas present in the mass analyzer as a collision gas.The radio-frequency (RF) amplitude, which had a substantialvoltage range, was set such that the peak height of the mole-cular ion decreased by at least 50%. The analyses were per-formed in both positive- and negative-ion mode.Accurate mass measurements were performed using a

Shimadzu high-speed liquid chromatography ion trap time-of-flight mass spectrometry system (LCMS-IT-TOF) equippedwith an ESI source. The conjugates were dissolved in chloro-form/methanol (1:1 v/v) and introduced into the ESI sourceby continuous infusion using the dosing pump for injectedsamples at a rate of 10 mL/min. The LCMS-IT-TOF ESI sourcewas operated at 4.5 kVand the capillary was heated to 200 �C.Nitrogen was used as the nebulizing gas. The MS/MS experi-ments were conducted in the collision cell using argon as thecollision gas.

RESULTS AND DISCUSSION

The results of anionic ring-opening oligomerization of b-butyrolactone initiated by lipoic acid potassium salt leading toconjugates of lipoic acid with oligo-3-hydroxybutyrate (LA-OHB) (outlined in Scheme 1) are summarized in Table 1. Theresults presented in Table 1 indicate that the oligomerizationreaction proceeded quantitatively and that oligomers havingmolecular weights close to those calculated were obtained.Taking into consideration the prospective use of synthesized

conjugates in cosmetics and their extensive contact with skin, itwas important to elucidate the structure and homogeneity of

Figure 1. 1H NMR spectrum (in CDCl3) ofoligomerization of b-butyrolactone initiated

wileyonlinelibrary.com/journal/rcm Copyright © 2013 John Wi

the product. NMR was used to obtain structural informationabout the synthesized conjugates. The 1H NMR spectrum ofthe oligomers obtained via ROO of b-butyrolactone initiated byLA potassium salt showed that, in addition to signals corre-sponding to protons of 3-hydroxybutyrate repeating units (Peaks1, 2, and 3), signals characteristic of the protons of (R)-5-(1,2-dithiolan-3-yl)pentanoate (originating from initiator incorpo-rated into the OHB chain) end group were present (Peaks 4–12,Fig. 1). Moreover, the signals at d=1.80 as well as those ofvery low intensity at 5.80 and 6.95 ppm can be attributed tothe protons of the crotonate end groups CH3-CH=CH (15),CH3-CH=CH (14), and CH3-CH=CH (13), respectively. Thecontent (in mol%) of minor products of oligo-3-hydroxybutyrateterminated by crotonate and carboxyl groups was estimatedfrom the1HNMR spectrum (Fig. 1) based on the ratio of integralsof the signal corresponding to crotonate end groups presentin theminor product and the (R)-5-(1,2-dithiolan-3-yl)pentanoateend group present in the conjugate. The estimated contentwas 4.6–4.8 mol%, depending on the signals selected forcalculation.

Molecular structural studies of lipoic acid–oligo(3-hydroxybutyrate)conjugates

To obtain detailed information about the structure at themolecular level of the LA-OHB conjugates obtained in thisstudy, electrospray ion trap multistage mass spectrometry(ESI-MSn) has been applied.

The ESI mass spectrum of oligomers obtained by ring-opening oligomerization of racemic b-butyrolactone initiatedby lipoic acid potassium salt (Sample 3, Table 1) is shown inFig. 2. Three series of singly charged ions with a peak-to-peakmass increment of 86 Da, equal to the molecular weight of the3-hydroxybutyrate repeating unit, were observed in the massspectrum (Fig. 2). The structures of the end groups and

oligomers obtained via the ring-openingby lipoic acid potassium salt.

ley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2013, 27, 773–783

Figure 2. (a) ESI mass spectrum (positive-ion mode) of oligomers obtained via the ring-opening oligomerization of b-butyrolactone initiated by lipoic acid potassium salt(Sample 3, Table 1). (b) Spectral expansion in the mass range m/z 1080–1240.

Scheme 2. Chemical structures of the ions shown in the massspectra of oligomers obtained via the ring-opening oligomer-ization of b-butyrolactone initiated by lipoic acid potassiumsalt (Figs. 2(a) and 2(b)).

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repeating units can be inferred from the masses of the singlycharged ions observed in the mass spectrum. The series ofions labeled as Series A (Figs. 2(a) and 2(b)), correspond tothe sodium-cationized LA-OHB conjugate chains terminatedby (R)-5-(1,2-dithiolan-3-yl)pentanoate (derived from theinitiator) and carboxyl end groups. Series A’ corresponds tothe sodium-cationized LA-OHB conjugate in the form of thesodium salt. The third series of ions (with very low relativeintensity and located especially in the low mass range),labeled as Series B (see Figs. 2(a) and 2(b)), can be assignedto the sodium-cationized oligo(3-hydroxybutyrate) chainsterminated by crotonate and carboxyl end groups. Chemicalstructures of the ions (Series A, A’ and B) appearing in themass spectrum (Fig. 2) are depicted in Scheme 2.Thus, the ions at m/z 1089 and 1175 observed in the

expanded region of the mass spectrum (Fig. 2(a)) were identi-fied as sodium adducts of LA-OHB conjugate chains contain-ing ten and eleven 3-hydroxybutyrate repeating units. Thesignals atm/z 1141 and 1227 were assigned to sodium adductsof oligo(3-hydroxybutyrate) with crotonate end groups con-taining thirteen and fourteen 3-hydroxybutyrate repeatingunits, respectively.

Copyright © 2013 JRapid Commun. Mass Spectrom. 2013, 27, 773–783

Based on the intensities of the series of ions correspondingto the conjugate macromolecules (Series A) and series of ionscorresponding to oligo-3-hydroxybutyrate terminated bycrotonate and carboxyl end groups (Series B), the content ofthe minor Series B was estimated to be equal to ~5%.

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The ions corresponding to the Series A and A’ present inthe mass spectrum (Figs. 2(a) and 2(b)) were also distin-guished by their characteristic isotopic profiles. The charac-teristic profile of conjugate molecular ions results from thepresence of sulfur atoms, which have four stable isotopes:32S (95.02%), 33S (0.75%), 34S (4.21%), and 36S (0.02%).To confirm that the main series of ions present in the mass

spectrum (Fig. 2) corresponded to LA-OHB conjugate chains,the theoretical isotopic profile for the selected ion of LA-OHBwith the assumed structure was calculated and comparedwith the experimentally obtained isotopic profile (Fig. 3).The theoretical isotopic profile for molecular ions of LA-OHB was calculated using the Isotope Distribution Calculatorand Mass Spec Plotter software available online.[30] As anexample, the calculated and experimental isotopic profilesfor the selected molecular ion of LA-(OHB)8 conjugate atm/z 917 are compared in Fig. 3.A deeper insight into the structure of individual conjugate

chains was obtained by the application of tandem massspectrometry (ESI-MS/MS). To verify the structural assign-ment of the individual conjugate chains, ESI-MS/MSfragmentation experiments of ions selected from Series Awereperformed.

Figure 3. Comparison of the experimeprofiles for the sodium adduct of the LA

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Previously conducted fragmentation studies of individualmolecular ions of polyesters (derivatives of b-hydroxyacids) revealed that b-hydrogen rearrangement was themain mechanism inducing fragmentation of such polyestersby ester bond cleavages.[31–36] Figure 4(a) shows the ESI-MS/MS spectrum of the precursor ion atm/z 1089 correspond-ing to the sodium adduct of LA-OHB conjugate chainscontaining ten 3-hydroxybutyrate units (Series A, Sample 3,Table 1).

The fragmentation of this precursor ion, which resulted fromthe random breakage of ester bonds along the oligomer chain,led to the formation of two series of product ions. According tothe structures assigned, the product ions at m/z 1003, 917, 831,745, 659, and 573 correspond to 3-hydroxybutyrate oligomerscontaining carboxyl and (R)-5-(1,2-dithiolan-3-yl)pentanoateend groups (originating from LA incorporated into the OHBchain). The complementary product ions at m/z 883, 797, 711,625, 539, and 453 correspond to oligo-3-hydroxybutyrate chainsterminated by crotonate and carboxylic acid groups. Thus, theproduct ion at m/z 883 corresponds to the oligomer formed bythe loss of lipoic acid molecule (206 Da), and the product ion atm/z 1003 corresponds to the oligomer formed by the loss ofcrotonic acid (86 Da; see Fig. 4(a) and Scheme 3).

ntal (a) and theoretical (b) isotopic-(OHB)8 conjugate at m/z 917.

ley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2013, 27, 773–783

Figure 4. ESI-MS/MS product ion spectra (positive-ionmode) of the selected sodium adduct of (a) the LA-OHBconjugate at m/z 1089 and (b) oligo-3-hydroxybutyrate termi-nated by crotonate and carboxyl end groups.

Scheme 3. Fragmentation pathway of the sodium adduct ofthe LA-OHB conjugate ion at m/z 1089.

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Figure 4(b) shows the ESI-MS/MS product ions spectrumof the sodium adduct of the oligo(3-hydroxybutyrate) ion atm/z 1141 containing crotonate and carboxyl end groupsselected from Series B (Figs. 2(a) and 2(b)). In this case, thebreakage of the ester bonds along the oligomer led to the for-mation of only one set of oligo(3-hydroxybutyrate) productions terminated by carboxyl and crotonate end groups (seefragmentation pathway in Fig. 4(b)).

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The product ion at m/z 1055 was formed by the loss ofcrotonic acid from both ends of the precursor ion at m/z1141. The fragmentation of sodium adduct ions of oligo-3-hydroxybutyrate with crotonate end groups has been pre-viously reported.[32,37]

The comparison of the fragmentation spectra shown inFig. 4(a) with the respective theoretical fragmentation pathwaysclearly indicates that Series A corresponds to the 3-hydroxybuty-rate oligomers containing (R)-5-(1,2-dithiolan-3-yl)pentanoateand carboxyl end groups (Fig. 4(a)). The minor Series B wasattributed to 3-hydroxybutyrate oligomers with crotonate andcarboxyl end groups (Fig. 4(b)).

The formation of unsaturated (crotonate) end groups inb-butyrolactone polymerization,[12,37,38] as well as 4-ethoxy-2-butenoate end groups in the polymerization of b-(ethoxy-methyl)-b-propiolactone initiated with activated carboxylic acidanions, has been reported.[36] Likewise, the formation of unsatu-rated end groups in the anionic ring-opening polymerization ofb-butyrolactone initiated by activated LA potassium salt maybe caused by a chain-transfer reaction to the monomer and/oran intermolecular carboxylate-induced a-deprotonation.

To confirm the structural assignments for the observedfragmentation patterns in the ESI-MS/MS product ions spec-trum shown in Fig. 4(a), accurate mass measurements weremade using the ESI-MS-IT-TOF spectrometer. The production mass spectrum obtained for the selected sodium adductsof the LA-OHB conjugate at m/z 659.2 is shown in Fig. 5, andthe accurate mass data and assignment of product ions aresummarized in Table 2.

Cytotoxicity of LA-OHB conjugates

In vitro cytotoxicity tests demonstrated the effects of lipoicacid and its conjugates with oligo-3-hydroxybutyrate(Samples 1–4, Table 1) on keratinocytes (epidermoid carci-noma cell line KB). The results of the MTT cytotoxicity assayperformed on two LA-OHB conjugates of different molecularweights are shown in Fig. 6 (Sample 2 was entirely soluble inwater, and Sample 4 was non-soluble in water). This experi-ment was conducted to confirm the biocompatibility of theconjugates, which is necessary for future applications incosmetics.

After exposure to the two conjugates, only small differ-ences in cell viability were observed. LA did not affect cellviability, nor did its conjugates have cytotoxic effects. Oneof the LA-OHB conjugates (Sample 2, Table 1) appeared tohave more potential for cosmetic use as it stimulated cellviability at the lowest concentration (1 mg/mL; Fig. 6);however, this result was not statistically significant. Therefore,further tests and more detailed analysis are needed to charac-terize the stimulation of cell proliferation by LA-OHB conju-gates and their penetration through the stratum corneum.

ESI-MS analysis of water-soluble hydrolytic degradation productsof LA-OHB conjugates

Considering the prospective application of the synthesizedconjugates as controlled release systems for bioactive sub-stance in cosmetics, preliminary studies of their hydrolyticdegradation as well as the release of lipoic acid were per-formed. ESI-MS was used to identify released a-lipoic acidand oligomeric water-soluble degradation products formed

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Figure 5. ESI-MS/MS product ion spectrum of selected sodium adduct of theLA-OHB conjugate at m/z 659.2.

Table 2. Accurate mass data and assignment of product ions arising from the fragmentation of sodium adduct of LA-OHBconjugate at m/z 659.2.

Assignment Measured m/z Theoretical m/z Error (ppm) Elemental composition

B3 281.1035 281.1001 12.0 C12H18NaO6+

A1 315.0709 315.0701 2.5 C12H20NaO4S2+

B4 367.1397 367.1369 7.6 C16H24Na08+

A2 401.1103 401.1069 8.4 C16H26NaO6S2+

B5 453.1757 453.1737 4.4 C20H30Na010+

A3 487.1474 487.1436 7.8 C20H32NaO8S2+

A4 573.1839 573.1804 6.1 C24H38NaO10S2+

[M+Na]+ I.S. 659.2172 C28H44NaO12S2+

I.S.: internal standard – precursor ion i.e. sodium adduct of LA-(OHB)5 conjugate:

HOO

OCH3O

SS

++Na

5

m/z value of I.S. has been calculated using Accurate Mass Calculator which was provided with the Shimadzu high-speedliquid chromatograph mass spectrometer (LCMS-IT-TOF) software

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during the hydrolytic degradation process of selected LA-OHB conjugates. The analysis of water-soluble degradationproducts was performed in negative-ion mode, which madeit easier to simultaneously monitor the signals correspondingto released lipoic acid and oligomeric degradation products.Figure 7 shows negative ESI-MS spectra of solutions before

(Fig. 7(a)) and after (Fig. 7(b)) 7 days of incubation of the LA-OHB conjugate in water at 37 �C. Expansions of the massrange m/z 540–820 are shown in Figs. 8(a) and 8(b). One mainset of anions with a peak-to-peak mass increment of 86 Da(labeled as Series A) was observed in the mass spectra (Figs.7(a) and 8(a)). The signals belonging to Series A correspondto oligo-3-hydroxybutyrate chains terminated by lipoic acidand carboxylic end groups. Another series with very low inten-sity (labeled as Series B) observed in these spectra corresponded

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to oligo-3-hydroxybutyrate chains with crotonate and carbox-ylate end groups. Additionally, in the mass spectra shown inFigs. 7(b) and 8(b), a third series of anions, labeled as Series C,was identified. These signals correspond to oligo-3-hydroxy-butyrate chains terminated by hydroxyl and carboxylate endgroups, which were formed by hydrolysis of OHB chains.The general chemical structures of the identified oligomers(Series A, B, and C) are shown in Scheme 4.

The mass spectra shown in Figs. 7 and 8 reveal the changesin the distribution of the ion patterns of the LA-OHB conju-gate before and after 7 days of hydrolytic degradation. Themass spectrum acquired directly after dissolving the LA-OHB conjugate in water shows a typical distributionobserved for a sample with low dispersity (Fig. 7(a)), whereasthe spectrum recorded after 7 days of hydrolysis shows that

ley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2013, 27, 773–783

Figure 6. Cytotoxicity of LA-OHB conjugates assessed byMTT assay. KB cells were treated with lipoic acid and LA-OHB conjugates (Samples 2 and 4, Table 1) as described inMTT cytotoxicity assay; n = 6, means� SD; P <0.05.

Figure 7. ESI mass spectra (negative-ion mode) of (a) watersolution of LA-OHB conjugate before incubation at 37 �C(Sample 2, Table 1) and (b) water-soluble degradationproducts formed after 7 days of hydrolytic degradation ofthe LA-OHB conjugate at 37 �C.

Figure 8. Expanded region m/z 540–810 of the mass spectrashown in Fig. 7 of (a) water solution of LA-OHB conjugatebefore incubation at 37 �C (Sample 2, Table 1) and (b) water-soluble degradation products formed after 7 days of hydroly-tic degradation of the LA-OHB conjugate at 37 �C.

ESI-MS structural studies on LA-OHB conjugates

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intensity of the signals corresponding to oligomers withlower molecular weights increased as a function of hydrolysistime (Fig. 7(b)). The shift of the main series of signals to lowerm/z values indicates the formation of shorter LA-OHBconjugate chains, particularly those corresponding to conju-gate chains containing one and two 3-hydroxybutyrate units(signals atm/z 291 and 377; Fig. 7(b)). Additionally, the forma-tion of oligo-3-hydroxybutyrate terminated by hydroxyl andcarboxylate end groups (Series C) indicates that the hydroly-tic degradation process progressed over the entire 7-dayperiod. Moreover, a high intensity signal corresponding tofree a-lipoic acid (m/z 205) was observed, confirming therelease of a-lipoic acid from the conjugate chain into solution.It is known that when applying ESI-MS to mixtures con-

taining both acids and oligomers, the intensities of the signalscorresponding to acids are lower than those of theoligomers.[39] The quantitative estimation of the oligomerand acid contents would require separate calibrations foreach component type. However, the results we obtainedconfirmed both the release of lipoic acid and the formationof shorter chains of LA-OHB conjugate as well as oligo-3-hydroxybutyrate terminated by hydroxyl and carboxylate

wileyonlinelibrary.com/journal/rcmCopyright © 2013 John Wiley & Sons, Ltd.Rapid Commun. Mass Spectrom. 2013, 27, 773–783

1

Scheme 4. Chemical structures of the ions shown in thenegative ESI mass spectra of water-soluble degradationproducts of the LA-OHB conjugate (Figs. 7 and 8).

Scheme 5. Hydrolytic degradation pathway of the LA-OHBconjugate.

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end groups. Thus, the results of the mass spectrometry analy-sis indicated that the hydrolysis of ester bonds occurred in arandom manner along the LA-OHB conjugate chains (asshown in Scheme 5).Studies on release rate and controlled release profiles of

LA-OHB delivery systems are underway in our laboratory.

CONCLUSIONS

The results of the present study revealed that anionic ROO ofb-butyrolactone initiated by potassium salt of lipoic acid ledto the formation of a conjugate of 3-hydroxybutyrate withlipoic acid. Based on mass spectrometry and NMR results, itwas found that carboxylate anions of lipoic acid potassiumsalt (activated by highly polar DMSO employed as a solvent)opened the alkyl–oxygen bonds of the (R,S)-b-butyrolactonevia the formation of oligo-3-hydroxybutyrate bearing car-boxylic and (R)-5-(1,2-dithiolan-3-yl)pentanoate end groups.The ESI-MS/MS fragmentation experiments performed forselected conjugate precursor ions confirmed that the initiatormoiety, (R)-5-(1,2-dithiolan-3-yl)pentanoate, was covalentlybonded with the oligo-3-hydroxybutyrate chain.Preliminary comparative cytotoxicity studies showed that

the synthesized conjugates were non-toxic, opening theirpotential application in cosmetics.Furthermore, application of mass spectrometry to monitor

the hydrolytic degradation process of the bioconjugates pro-vided detailed insight and allowed the identification of thedegradation products formed and the confirmation that a-lipoic acid was released from the conjugates into solution.

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AcknowledgementsThis work was supported by the European Regional Develop-ment Fund: Contract No. POIG.01.03.01-00-018/08 "Newgeneration packaging materials made from plastics subjectto the organic recycling" in the framework of the InnovativeEconomy Operational Programme (IE OP). M.M. is a scholar-ship holder within the DoktoRIS project-scholarship programfor the innovation of Silesia region supported by theEuropean Community from the European Social Fund.

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