Total Hydroxycinnamic Acids Assay: Prevalidationand Application on Lamiaceae Species

Maja Bival Štefan & Jadranka Vuković Rodríguez &

Biljana Blažeković & Marija Kindl &Sanda Vladimir-Knežević

Received: 21 January 2013 /Accepted: 18 April 2013# Springer Science+Business Media New York 2013

Abstract In this work, qualitative and quantitative analysesof total hydroxycinnamic acid (THA) derivatives in leaves,flowers and aerial parts of 32 Lamiaceae species were car-ried out. HPTLC analysis of plant material indicatedrosmarinic acid as the main phenolic acid in 22 Lamiaceaeplant extracts, while chlorogenic acid predominated in other10 investigated species. Determination of THA in differentmedicinal plants was performed by colorimetric methodsusing chromogenic system of HCl-NaNO2–Na2MoO4–NaOH and two standards: rosmarinic acid (R-THA proce-dure) and chlorogenic acid (C-THA procedure). Perfor-mance characteristics of THA assay were established, andthe quality of the analytical procedures was evaluated usingvaluable prevalidation strategy. Good quality of the mea-surements at the lower analyte level, excellent resolution ofblank and analyte signals, homogenous data material, ideallinear calibration and analytical evaluation functions, verylow limit of detection [LD(R-THA)=0.53 μg; LD(C-THD)=1.75 μg] and limit of quantitation [LQ(R-THD)=1.76 μg;LQ(C-THD)=4.21 μg], and high precision and accuracyconfirmed the high quality of investigated procedures asvaluable tools in THA analysis. Application of theseprevalidated, simple and rapid analytical methods revealedthat the studied Lamiaceae plants differ greatly in their THAcontents, ranging from 1.95 % (Acinos arvensis) to 11.03 %(Satureja subspicata). The results obtained from qualitativeand quantitative analysis provided new information

regarding phytochemical characterization of Lamiaceae spe-cies originating from Croatia and highlighted members ofthe genus Micromeria, Origanum, Melissa and Satureja asrich sources of hydroxycinnamic acids.

Keywords Total hydroxycinnamic acid derivatives . Phenolicacids . Prevalidation strategy . UV–Vis spectrophotometry .

Lamiaceae species

Abbreviationsx Analyte amountB Blank measurementy Standard measurementS Corrected signal (net signal)A Measure of particular sensitivitys Standard deviationsr Relative standard deviations Mean standard deviationsr Mean relative standard deviationAC Ratio between y6 and B6

s2Bw Dispersion of blanks within groupss2Bb Dispersion of blanks between groupsBN Grand blank meansBN Total standard deviation of blank signalssrBN Total relative standard deviation of blank signalsr2 Determination coefficientb Slope of a linea Intercept of a linesb Errors in the slopesa Errors in the intercept±Cb Confidence limit for the slope±Ca Confidence limit for the interceptV Constant of calibration functionV Constant of analytical evaluation functionsV Standard deviation of the constant V of calibration

function

M. B. Štefan :B. Blažeković :M. Kindl : S. Vladimir-KneževićDepartment of Pharmacognosy, Faculty of Pharmacy andBiochemistry, University of Zagreb, Marulićev trg 20,10000, Zagreb, Croatia

J. Vuković Rodríguez (*)Department of Analytics and Control of Medicines, Faculty ofPharmacy and Biochemistry, University of Zagreb, A. Kovačića 1,10000, Zagreb, Croatiae-mail: jvukovic@pharma.hr

Food Anal. MethodsDOI 10.1007/s12161-013-9630-8

sV Standard deviation of the constant V of analyticalevaluation function

sM Standard deviation of the analytical procedurebS Apparent signal valuesbx Apparent quantity of analyteS* Outlying value in the set of signalsx* Outlying value in the set of analyte quantitiesSD Limiting value for net signalLD Limit of detectionLQ Limit of quantitationΔx Difference between actual, x, and apparent, bx, values

Introduction

The Lamiaceae is one of the largest, most distinctive andmost diversified plant families, which includes about 236genera and over 7,100 species of flowering plants world-wide (Harley et al. 2004). Well-known for its many aromat-ic, medicinal and culinary plants, this cosmopolitan familyis especially abundant in the Mediterranean region. In theflora of Croatia, the Lamiaceae family is represented with atotal of 36 genera and a total of 160 species (Flora CroaticaDatabase 2004). Owing to their richness in essential oil,phenolic acids, flavonoids and other bioactive constituents,members of this family are attracting a growing scientificinterest due to a wide variety of potential applications inpharmaceutical, cosmetic and food industries. The plantspecies and/or their main constituents of high biologicalactivity can be applied either as herbal drugs or innutraceuticals, food supplements and functional food.

Phenolic acids are themost common naturally occurring plantpolyphenols comprising a diverse group of hydroxybenzoic andhydroxycinnamic acids and their derivatives. Although theymayexist in the free form, conjugated forms of phenolic acids pre-dominate in the plant, being linked through ester, ether or acetalbonds either to structural components of the plant, larger poly-phenols or smaller organic molecules (Vladimir-Knežević et al.2012). Rosmarinic acid (RA), an ester of caffeic acid and 3,4-dihydroxyphenyllactic acid, is a hydroxycinnamate most com-monly occurring in the species of Lamiaceae plant family. Thisphenolic acid is known to have a number of interesting biolog-ical effects, the main ones being anti-inflammatory, antioxidant,antiallergic, antibacterial, antiviral, antimutagenic andantidepressive (Petersen and Simmonds 2003; Takeda et al.2002; Osakabe et al. 2004). More recently, antiangiogenic,antitumor and antihepatotoxic effects of RA have also beenreported (Kim et al. 2009; Anusuya and Manoharan 2011;Domitrović et al. 2012). Chlorogenic acid (CA), which is formedby esterification of caffeic and quinic acid, is a hydroxycinnamicacid ester that is more widely distributed in the plant kingdom.CA has been reported to exhibit a wide range of biologicaleffects, including antioxidant, anti-inflammatory, anticancer,

anti-obesity, antimicrobial, antiviral and neuroprotective activi-ties (Belkaid et al. 2006; Kwon et al. 2010; Cho et al. 2010;Weng and Yen 2012).

The quantities of phenolic acids in plants are influenced bygenetically determined properties, localities, environmentalconditions, harvesting time as well as storage method(Fernandez-Orozco et al. 2010; Skrzypczak-Pietraszek andPietraszek 2012). For this reason, it is important to determinethe real level of these compounds present in plant materials ofdifferent origin. Nowadays, various techniques for determina-tion of phenolic acids in plant matrix are used, such as highperformance liquid chromatography (Robbins 2003; Irakli et al.2012), gas chromatography (Wang and Zuo 2011; León-González et al. 2013), capillary electrophoresis (Bonoli et al.2003; Lee et al. 2011) and UV–Vis spectrophotometry (Öztürket al. 2010; Vladimir-Knežević et al. 2011). Although it isincreasingly important to be able to recognize the differentstructures of these phenolic substances and to determine theirquantities, the establishment of a simple method that enablesquantification of the total amount of phenolic acids in plantmaterials is equally important. For this purpose, global andnonspecific methods are more appropriate, both from the per-spectives of time and cost. Among all techniques mentionedabove, UV/Vis spectrophotometry can be used to determine thetotal amount of different phenolic compounds in order toevaluate the quality of the raw plant material and to standardizephytopharmaceuticals (Zhu et al. 2010; Fuentes et al. 2012).

Analysis of bioactive substances and quality control ofherbal drugs as well as introduction of standardized analyt-ical methods in routine analysis require analytical proce-dures with good performance characteristics. For thisreason, complete prevalidation strategy (Grdinić andVuković 2004), as an informative screening method provedto be useful for preliminary evaluation of an analyticalprocess. The aim of prevalidation proposal based on peculiarapproaches is to diagnose the quality of an analytical procedureand to decide whether a method in question is capable ofproducing accurate and reliable data. The efficiency ofprevalidation procedure is given by characteristic data such asconstants of calibration and analytical evaluation function,standard deviation of procedure, limit of quantitation, precisionand accuracy, and other metrological characteristics.

The present study includes qualitative and quantitativeanalyses of hydroxycinnamic acids in 32 Lamiaceae speciesgrowing in Croatia. Preliminary qualitative phytochemicalanalysis aimed to reveal the presence and composition ofphenolic acids. Determination of total hydroxycinnamicacids (THA) in selected medicinal plants was performedby spectrophotometric method using chromogenic systemof HCl-NaNO2–Na2MoO4–NaOH which forms complexeswith ortho-dihydroxyphenols (EDQM 2011, pp. 1227 and1059; Vladimir-Knežević et al. 2012). Since two differentphenolic acids (RA and CA) are used in official methods as

Food Anal. Methods

standards against which the THA content is expressed, thepurpose of this study was to analyse the reactivity of RA andCA with the above-mentioned methods and estimate theirinfluence on the values obtained as well as ultimately evaluatethe quality of used colorimetric method. Therefore, prior to thequantitative analysis performance characteristics of THA de-rivatives, procedures (THA procedures) were established, andthe quality of the procedures was evaluated using valuableprevalidation strategy. These prevalidated analytical methodswere applied to provide a new data regarding hydroxycinnamicacids amounts of selected Lamiaceae species from Croatia inorder to find the richest sources of these potent bioactiveconstituents.

Materials and Methods

Apparatus

UV/Vis spectrophotometer Agilent 8453 with PC-HP 845xUV–Visible System (Agilent, Germany) and 1-cm quartzcells were used for all absorbance measurements.

Chemicals

Sodium hydroxide, sodium molybdate dihydrate, sodium ni-trite and hydrochloric acid were purchased from Kemika(Zagreb, Croatia). Chlorogenic acid (CA, ≥95 %) androsmarinic acid (RA, 96 %) were obtained from Sigma-Aldrich (St. Louis, MO, USA). 2-Aminoethyl diphenylborinate(NP), polyethylene glycol 4000 (PEG) and quercetin-3-rutinoside (rutin, ≥95 %) were purchased from Fluka (Buchs,Switzerland). Other reagents and solvents used were ofanalytical grade.

Plant Material

A total of 32 Lamiaceae species used in this study, listed inTable 10, were collected during the flowering period fromMay to September 2011 in Croatia. The plant species wereidentified at the Department of Pharmacognosy, Faculty ofPharmacy and Biochemistry, University of Zagreb, Croatia,where the voucher specimens have been deposited. Air-dried and pulverized leaves, flowers and aerial plant partswere phytochemically investigated.

HPTLC Analysis

High performance thin-layer chromatographic (HPTLC) anal-ysis of phenolic acids was performed on precoated silica gel60 F254 TLC plate (Merck, Germany). Aliquots (10 μL) of10 % plant methanolic extracts and 0.5 % methanolic solutionof reference substances were manually applied on the plates

which were then developed in vertical glass chambers previ-ously saturated with the mobile phase diisopropyl ether–acetone–water–formic acid (50:30:10:10, v/v/v/v). After de-velopment, the plate was dried in a stream of air for a fewminutes. Phenolic acids were detected under UV light at365 nm after spraying with natural products: polyethyleneglycol reagent (1 % methanolic solution of 2-aminoethyldiphenylborinate and 5 % ethanolic solution of PEG 4000,NP/PEG; Fecka and Turek 2008).

Prevalidation Protocol

The prevalidation experiments were based on the 24 measure-ments divided in six analytical groups (6 standard solutions ofanalyte, j). Each analytical group comprises four repeatedexperiments (i). For each measurement of the standard, themeasurements of corresponding blank solution (24 blankmeasurements) and compensation solution were performed(24 compensation measurements). To check the quality ofthe THA derivatives procedures (THA procedures), two stan-dards were used: RA (R-THA system) and CA (C-THAsystem). Analyte stock solutions of RA and CAwere preparedby diluting 10.0 mg of each analyte in water and diluting up to100.0 mL with the same solvent. Working standard solutionswere prepared by an appropriate dilution of the standard stocksolutions. Standards and blanks were measured in a standardworking range of one power of ten (1.0xU=x1=xU=400.0 μg,upper level of analyte, 0.8xU=x2=320.0 μg, 0.6xU=x3=240.0 μg, 0.4xU=x4=180.0 μg, 0.2xU=x5=80.0 μg, 0.1xU=x6=xL=40.0 μg, lower level of analyte), alternately in thefollowing group sequence: 1, 6, 2, 5, 3 and 4. Measurementswere performed according to the procedure described underthe section “THAAssay”. Blank solutions were prepared, andabsorbance was identically measured, but without the analyte.Compensation solutions contained the same quantity of RA(CA) and other components as corresponding analyte solutionexcept reagents. The data were analyzed using descriptive andprognostic statistics to assess the measurement quality at thelower end of the analytical working range, data homogeneity,determine calibration and analytical evaluation functions,detect outliers and estimate limiting values, precision andaccuracy. The complete prevalidation strategy with all re-quirements (R) and algorithms was described previously(Grdinić and Vuković 2004). Throughout the paper and inthe tables, statistical testing values are based on the Rsdefined in that work. All calculations were performedusing self-made computer program called ESKULAP(Grdinić and Vuković 2004).

THA Assay

Determination of THA was performed according to theprocedure described in European Pharmacopoeia (EDQM

Food Anal. Methods

2011, p. 1227). To 0.200 g of the powdered drug, 80 ml ofalcohol (50 %v/v) was added, and the mixture was boiled ina water-bath under a reflux condenser for 30 min. Aftercooling, the extract was filtered and the filter rinsed with10 ml of alcohol (50 %v/v). Combined filtrate and therinsings were diluted to 100.0 ml in a volumetric flask withalcohol (50 %v/v) and obtained solution served as stocksolution. Test solution was prepared by mixing 1.0 ml ofthe stock solution with 2 ml of 0.5 M hydrochloric acid,2 ml of a solution prepared by dissolving 10 g of sodiumnitrite and 10 g of sodium molybdate in 100 ml of water,then 2 ml of dilute sodium hydroxide solution (8.5 %) wasadded and final dilution of the sample solution to 10.0 mlwith water. Compensation solution was prepared by diluting1.0 ml of the stock solution to 10.0 ml with water. Theabsorbance of the test solution was measured immediatelyat 505 nm. The content of THA, expressed as RA, wascalculated from the expression: (per cent)=A×2.5/m, whereA is absorbance of the test solution at 505 nm and m is massof the examined sample in grams, taking the specific absor-bance of RA to be 400.

In order to determine THA content expressed as CA, thefollowing procedure described in European Pharmacopoeiawas performed (EDQM 2011, p. 1059). Briefly, 0.300 g ofthe powdered drug was extracted with 95 mL of alcohol(50 %v/v) by boiling in a water-bath under a reflux con-denser for 30 min. The cooled extract was filtered, the filterrinsed with 5 ml of alcohol (50 %v/v), and then the com-bined filtrate and rinsing were diluted in a volumetric flaskto 100.0 mL with alcohol (50 %v/v). To an aliquot of 1.0 mLof the test solution were added 2 mL of 0.5 M hydrochloricacid, 2 ml of a solution prepared by dissolving 10 g ofsodium nitrite and 10 g of sodium molybdate in 100 ml ofwater, 2 ml of dilute sodium hydroxide solution (8.5 %) anddiluted to 10.0 mL with water. The absorbance of the testsolution was measured immediately at 525 nm, using acompensation solution prepared by mixing 1.0 ml of thetest solution, 2 ml of 0.5 M hydrochloric acid and 2 ml ofdilute sodium hydroxide solution R and diluting to 10.0 mlwith water. The content of THA expressed as CA wascalculated from the expression: (per cent)=A×5.3/m, whereA is absorbance at 525 nm and m is mass of the test samplein grams, taking the specific absorbance of CA at 525 nm tobe 188. All analyses were performed in triplicate.

Results and Discussion

Qualitative Analysis of Hydroxycinnamic Acidsin Lamiaceae Species

The presence of phenolic acids in methanolic extracts ofleaves, flowers and aerial parts of various Lamiaceae species

was established using HPTLC (Fig. 1). Two phenolic acidswere identified as RA and CA, respectively, verified by theirintense blue fluorescent bands as shown in Fig. 1 (Rf values0.79 and 0.33, respectively). RA was found to be predom-inant hydroxycinnamic acid derivate in most of the testedplants. In methanolic extracts of Lamium purpureum,Marrubium incanum, Marrubium vulgare and Teucriummontanum, RA was not detected. HPTLC analysis revealedCA as the main phenolic acid in Acinos arvensis,Calamintha officinalis, Calamintha sylvatica, Lamiumalbum, L. purpureum and T. montanum (Fig. 1).

Analysis of Prevalidation Results

To determine the THA content in particular medicinal plantssuch as Rosmarinus officinalis L., Melissa officinalis L. andFraxinus excelsior L., the European Pharmacopoeia (EDQM2011, p. 1227) recommends two very similar procedures thatdiffer primarily in the standard used (rosmarinic or chlorogenicacid) and therefore in some procedural steps. A completeprevalidation strategy was carried out to evaluate the analyticalparameters and quality of both THA procedures. Our objectivewas to examine how the choice of standard affects theprevalidation results and thereby provide guidance for selectingappropriate standards for determining the THA content ofmedicinal plants. Initial prevalidation data were as follows:amount of RA (CA) (x) were within the working range from40.0 to 400.0 μg, absorbances were obtained from measure-ments of the blank (B), standard (y) and the compensationsolution. The corrected absorbance (S) was calculated as thedifference y−B. Because of possible influence of RA (CA) onabsorbance of the complex, prior to mathematical/statisticaltesting, gross signal is corrected with the absorbance obtainedin the measurement of corresponding compensation solution.The system of diagnosis was used for the result analysisobtained in each prevalidation step.

The first step in descriptive statistics of the prevalidationprocess was to characterize six analytical groups of two in-vestigated systems (R-THA and C-THA systems). This char-acterization included calculation of average values, standardand relative standard deviations, as well as mean s and sr of allgroups (Tables 1 and 2). These values were used to evaluaterepeatability of the measurements as a part of precision(Thompson et al. 2002; IUPAC 1997). Precision very oftenvaries with analyte concentration; the standard deviation in-creases with concentration, whereas the relative standard de-viation decreases with concentration (Thompson 1988).Similarly, the acceptance criteria for precision depend verymuch on the type of analysis (concentration of analyte andanalytical technique; Huber 2001). Relative standard de-viations of blank absorbances showed low precision ofblank measurements in both systems. Nevertheless, thefluctuations in blank values could be ignored because

Food Anal. Methods

Fig. 1 a, b HPTLC chromatogram of phenolic acids in methanolicextracts of different Lamiaceae species originating from Croatia. Exper-imental conditions: HPTLC silica gel 60 F254 plate; diisopropyl ether–acetone–water–formic acid 5:3:1:1 (v/v/v/v) as mobile phase, NP/PEG for

detection, UV 365 nm. T1–3: rutin (Rf=0.26), chlorogenic acid (Rf=0.33)and rosmarinic acid (Rf=0.79) as references; for plant extracts, seeabbreviations in Table 10

Table 1 Characterization of groups 1–6 of R-THA system

Groupa (j) xb (μg) B sB srB %ð Þ== y sy sry %ð Þ��S ss srs %ð Þ== A

csA srA %ð Þ==

1 400 0.0010/±0.00005/±4.88 1.327/±0.015/±1.15 1.325/±0.015/±1.15 0.0033/±0.00004/±1.15

2 320 0.0013/±0.00005/±4.88 1.057/±0.010/±0.95 1.055/±0.010/±0.95 0.0033/±0.00003/±0.95

3 240 0.0010/±0.00005/±4.88 0.788/±0.008/±1.01 0.786/±0.008/±1.01 0.0033/±0.00003/±1.01

4 160 0.0010/±0.00005/±4.88 0.547/±0.010/±1.74 0.546/±0.010/±1.75 0.0034/±0.00006/±1.75

5 80 0.0010/±0.00005/±4.88 0.267/±0.004/±1.58 0.265/±0.004/±1.58 0.0033/±0.00005/±1.58

6 40 0.0023/±0.0010/±42.55 0.139/±0.003/±2.12 0.137/±0.003/±1.83 0.0034/±0.00006/±1.83

s sr;%ð Þ ±0.0004 (±17.93) ±0.009 (±1.49) ±0.009 (±1.42) ±0.00004 (±1.42)

a Total number of measurements: n=72, comprising 24 each of blanks, standards and compensation solutionsb Amount of rosmarinic acidcMeasure of particular sensitivity, An=Sn/xn

Food Anal. Methods

they were so small compared to the analyte signal.However, relative deviations of sample measurementsand corrected absorbances showed high precision forthe system with RA (sry from ±0.95 % to ±2.12 %,srS from ±0.95 % to ±1.83 %) and for the system withCA (sry from ±0.56% to ±2.86%, srS from ±0.55% to ±2.95%).Both systems satisfied the strict prevalidation criterionof sr<±5 % for precision (Grdinić and Vuković 2004).

The first step in prognostic mathematical/statistical eval-uation of data is a preliminary check of analytical groups 1and 6, which limit the analyte working range (Table 3, R1).Special emphasis was placed on quality control of measure-ments in the group with the smallest amount of RA (CA), x6,because accidental fluctuations of the blank measurementscould seriously affect reliability of the analytical system. Inthe R-THA and C-THA procedures, the blank signal issignificantly lower than the corrected signal in the groupwith x6. Although the blank deviations are high, the influ-ence of blank dispersions on standard deviation of the R-

THA and C-THA procedures is not expected because blanksignals were small in comparison to analyte signals (Table 3,R2). Furthermore, standard deviations of analyte andcorrected signals below ±2.5 % at the upper analyte leveland below ±25 % at the lower analyte level suggested thatdetermination limit is expected below x6. Additionalchecking of how well the systems resolved analyte andblank signals at x6 showed excellent resolution in bothsystems (Table 3, R4).

The homogeneity check was carried out using simple anal-ysis of variance (ANOVA) and Bartlett's test (Table 4).ANOVA (Massart et al. 1997; Wernimont 1985) applied tothe variances of the blank values indicated inhomogeneousblank values in R-THA (R=6.46) and C-THA (R=6.80) sys-tems. Additional checking (Table 4, R7) demonstrated againthat the influence of the blank in both systems could beconsidered negligible because they are small compared tocorresponding analyte signals at x1. According to theprevalidation criterion, the total sr values for blanks should

Table 2 Characterization of groups 1–6 of C-THA system

Groupa (j) xb (μg) B sB srB %ð Þ== y sy sry�

%ð Þ�S ss srS== %ð Þ A

csA srA %ð Þ==

1 400 0.0028/±0.0005/±18.2 0.816/±0.005/±0.56 0.814/±0.005/±0.55 0.0020/±0.00001/±0.55

2 320 0.0013/±0.0005/±40.0 0.677/±0.012/±1.87 0.676/±0.013/±1.85 0.0021/±0.00004/±1.85

3 240 0.0028/±0.0005/±18.2 0.499/±0.011/±2.19 0.496/±0.011/±2.12 0.0021/±0.00004/±2.12

4 160 0.0015/±0.0006/±38.5 0.329/±0.008/±2.28 0.327/±0.007/±2.25 0.0020/±0.00005/±2.25

5 80 0.0028/±0.0005/±18.2 0.163/±0.005/±2.86 0.160/±0.005/±2.95 0.0020/±0.00006/±2.95

6 40 0.0030/±0.0008/±27.2 0.083/±0.002/±2.10 0.080/±0.002/±2.62 0.0020/±0.00005/±2.62

s sr;%ð Þ ±0.0006 (±28.3) ±0.008 (±2.10) ±0.008 (±2.19) ±0.044 (±2.19)

a Total number of measurements: n=72, comprising 24 each of blanks, standards and compensation solutionsb Amount of chlorogenic acidcMeasure of particular sensitivity, An=Sn/xn

Table 3 Checking of limitinggroups 1 and 6

aDetailed requirements are de-fined in Grdinić and Vuković(2004)

Req.a Result Diagnosis

R-THA C-THA

R1 AC=61.8 AC=27.5 Blank signal is lower than the signal at lower analyte level, xLR2 R=607.8 % R=265.0 % Significant influence of blank dispersions on sM is not expected

srB1=±4.9 % srB1=±18.2 %

srB6=±42.6 % srB6=±27.2 %

R3 sry6=±2.12 % sry6=±2.10 % Determination limit is expected below x6srS6=±1.83 % srS6=±2.62 %

sry1=±1.15 % sry1=±0.56 %

srS1=±1.15 % srS1=±0.55 %

sr6<25 % sr6<25 %

sr1<2.5 % sr1<2.5 %

R4 R=35.1 R=31.1 Excellent resolution of signals

R5 R=2.87 R=1.74 Linear calibration function is expected

Food Anal. Methods

be lower than 50 % (Table 4, R8). Although the dis-persion of blank values in both systems was high, theirsmall values should not have serious influence on thequality of the measurements. Since these auxiliarycriteria were satisfied in both systems, each y value

was corrected with the grand blank mean (BN). To test

the homogeneity of s and srvalues for y, S, A andapparent mass (bx) values for the different groups, theBartlett's test was used. The test indicated that mostvalues of s and sr in both systems were strongly homo-geneous. Inhomogeneous s and sr values were obtainedonly for blank values in R-THA system (Table 4, R9).

Table 4 Homogeneity testing

sh strongly homogeneous, ihinhomogeneousaDetailed requirements are definedin Grdinić and Vuković (2004)

Req.a Result Diagnosis

R-THA C-THA

R6 s2Bw=1.00×10−6 s2Bw=2.27×10

−6 Inhomogeneous blank valuess2Bb =1.55×10

−7 s2Bb =3.33×10−7

R=6.46 R=6.80

R7 BN<0.0066 BN<0.0041 Influence of blank value is negligibleBN=0.0012 BN=0.0023

R8 srBN=47.4 % srBN=37.2 %sBN=5.82×10

−4 sBN=8.68×10−4

R9 R(sB)=50.1, ih R(sB)=1.22, shR(srB)=30.0, ih R(srB)=3.82, sh

R(sy)=7.83, sh R(sy)=10.11, sh

R(sry)=2.77, sh R(sry)=5.30, sh

R(ss)=8.65, sh R(ss)=8.94, sh

R(srS)=2.10, sh R(srS)=5.73, sh

R(sA)=2.34, sh R(sA)=5.56, sh

R(srA)=2.10, sh R(srA)=5.73, sh

Table 5 Quality of analyte-signal relationship

aDetailed requirements are de-fined in Grdinić and Vuković(2004)

Req.a Result Diagnosis

R-THA C-THA

R10 r2=0.9997 r2=0.9992b=0.0033 b=0.0021

a=0.0063 a=−0.0029

sy=±0.0031 sy=±0.0027

sb=±0.0009 sb=±0.00008

sa=±0.0012 sa=±0.0011

Centroid=(206.7, 0.686) Centroid=(206.7, 0.425)

R11 R=182.3 R=116.3 Correlation significant

R12 ±Cb=0.0033±0.0003 ±Cb=0.0021±0.0002±Ca=0.0063±0.0031 ±Ca=−0.0029±0.0030

t testing for reality of calibration constants

R13 V=0.0033 V=0.0021 Ideal calibration functionRV=342.7 RV=223.4

sV=±0.00001 sV=±0.00001

sM=±0.012 sM=±0.0110bS=0.0033x bS=0.0021x

t testing for reality of analytical evaluation constants

R14 V=302.0 V=484.9 Ideal analytical evaluation functionRV=342.7 RV=223.4

sV=0.88 sV=2.17

sM=3.47 sM=5.32

bx=302.0S bx=484.9S

Food Anal. Methods

The relationship between analytical signal (absorbance)and analyte content (mass) of RA (CA) was determinedusing three methods: preliminary linearity check, methodof least squares and systematic t testing of the reality ofconstants. As can be seen in Table 3, a preliminary linearitycheck applied to particular sensitivities (Avalues) of limitinggroups 1 and 6 showed that linear calibration function wasexpected in R-THA (R5=2.87) and C-THA (R5=1.74) sys-tems. The linearity of the R-THA procedure evaluated bythe method of least squares (Table 5) showed that linearrelationship of the R-THA procedure is described by equa-tion A=0.0033x+0.0063 and determination coefficient, r2=0.9997. Somewhat lower sensitivity was obtained for C-THA system (A=0.0021x−0.0029; r2=0.9992). Thoroughsystematic evaluation of analytical functions, constants weredetermined for the calibration and analytical evaluationfunctions over the entire analyte working range, and idealanalytical functions were found for both systems (Table 5,R13, R14). These analytical functions were used to testoutliers, and evaluate limiting values, apparent signal values

(bS) and apparent quantities of RA (CA) (bx).Outlier testing was performed to check whether any mea-

surement differed significantly from the others in the set ofsignals (S) used to carry out calibration function and in the setof RA (CA) quantity (x) used to evaluate analytical evaluationfunction. The presence of regression outliers in prevalidationprocess was checked by comparing |S*|and |x*|values with thet values of the P=95 % and 99 % confidence intervals. Oneoutlying value is detected in both systems, which is in accor-dance with prevalidation criterion (Table 6, R15).

Limiting values, such as limiting signal value, limit ofdetection and limit of quantification, were estimated using

analytical evaluation function and recommended conceptsof limiting values and presented in Table 7 (Thompson et al.2002; ICH 1996). For both systems, estimated limitingvalues were significantly lower than the amount of RA(CA) at the lowest level of analyte (<40 μg). These resultssuggested that very low amount of CA and even loweramount of RA could be determined using these methods.

The final calibration and analytical evaluation functions

were used to determine the apparent signal values (bS ) andapparent quantities of RA (CA) (bx ) (Tables 8 and 9).Information on accuracy was further obtained by comparingactual (x) and found (bx) amounts of analyte. The range ofrandom deviations, as a measure of precision of the R-THAsystem, was from ±0.95 % to ±1.82 %. Systematic devia-tions, as a measure of accuracy, were from −1.05 % to +3.23 % (Table 8). The C-THA system is also characterizedwith high precision ( srx from ±0.55 % to ±2.95 %) andaccuracy (Δx from −3.62 % to +2.36 %, Table 9).

Good quality of the measurements at the lower analytelevel, excellent resolution of blank and analyte signals,mostly homogenous data material, ideal linear calibrationand analytical evaluation functions, very good agreement ofactual (x) and found (bx ) amounts of RA (CA), very lowlimiting values and high precision and accuracy confirmedthe high quality of the R-THA and C-THA systems, andcould be successfully applied to the determination of RAand CA in plant material. Somewhat higher sensitivity of R-THA system is shown as an advantage of THA procedure

Table 6 Outlier testing

aDetailed requirements are de-fined in Grdinić and Vuković(2004)

Req.a Result Diagnosis

R-THA C-THA

R15 S*13 =2.46 S*5 =2.50 One outlying value, no objectionon data material2.807> S*13

�� ��>2.069 2.807> S*5�� ��>2.069

x*13 =2.45 x*5 =2.47 One outlying value, no objectionon data material2.807> x*13

�� ��>2.069 2.807> x*5�� ��>2.069

Table 7 Evaluation of limiting values

Req.a Result Diagnosis

R-THA C-THA

R16 SD=0.0022 SD=0.0038 SD<S6LD=0.53 μg LD=1.75 μg LD<x6LQ=1.76 μg LQ=4.21 μg LQ<x6

a Detailed requirements are defined in Grdinić and Vuković (2004)

Table 8 Metrological characteristics of R-THA system

Amount of RA (μg)

Actual 400.0 320.0 240.0 160.0 80.0 40.0

Found 400.2 318.7 237.5 164.9 80.2 41.3

Random deviations

sbx (μg) ±4.61 ±3.03 ±2.41 ±2.89 ±1.27 ±0.75

srbx (%) ±1.15 ±0.95 ±1.01 ±1.75 ±1.58 ±1.82

Systematic deviations

Δx (μg) +0.24 −1.29 −2.52 +4.93 +0.16 +1.29

Δx (%) +0.06 −0.40 −1.05 +3.09 +0.20 +3.23

Six groups mean for bx: s=±2.79; sr =±1.42 %

Bartlett's test for bx: R(s)=8.65, sh; R(sr)=2.10, sh

Food Anal. Methods

where RA is used as a standard. One of the disadvantages ofthe procedures is high dispersions of the blank values.However, since their values are small compared to theanalyte values, their influence on the quality of the pro-cedures is not expected.

Quantitative Analysis of Hydroxycinnamic Acidsin Lamiaceae Species

The contents of THA in the 32 medicinal plants ofLamiaceae were determined spectrophotometrically, andobtained results are shown in Table 10. Considering thepreliminary results of HPTLC analysis, which indicated thatRA is the main phenolic acid in 22 Lamiaceae plant extracts

Table 9 Metrological characteristics of C-THA system

Amount of CA (μg)

Actual 400.0 320.0 240.0 160.0 80.0 40.0

Found 394.5 327.6 240.4 158.7 77.5 38.6

Random deviations

sbx (μg) ±2.17 ±6.06 ±5.09 ±3.57 ±2.29 ±1.01

srbx (%) ±0.55 ±1.85 ±2.12 ±2.25 ±2.95 ±2.62

Systematic deviations

Δx (μg) −5.52 +7.56 +0.40 −1.31 −2.53 −1.45

Δx (%) −1.38 +2.36 +0.17 −0.82 −3.17 −3.62

Six groups mean for bx: s=±3.80; sr =±2.19 %

Bartlett's test for bx: R(s)=8.94, sh; R(sr)=5.73, sh

Table 10 Contents of THA inLamiaceae species originatingfrom Croatia

aResults expressed as average ±standard deviation (SD) of threeindependent assays

Plant species Abbr. Plant part Content (%)±SDa

RA CA

Acinos arvensis (Lam.) Dandy Aa Aerial 1.95±0.15

Calamintha grandiflora (L.) Moench Cg Aerial 3.99±0.03

Calamintha officinalis Moench Co Aerial 4.01±0.16

Calamintha sylvatica Bromf. Cs Aerial 7.13±0.11

Hyssopus officinalis L. Ho Flower 3.59±0.06

Lamium album L. La Flower 6.40±0.34

Lamium purpureum L. Lp Flower 3.56±0.05

Lavandula angustifolia Mill. Lan Flower 2.82±0.07

Lavandula x intermedia Emeric ex Lois. Li Flower 4.17±0.04

Marrubium incanum Desr. Mi Aerial 2.08±0.05

Marrubium vulgare L. Mv Aerial 2.21±0.10

Melissa officinalis L. Mo Leaf 9.86±0.05

Mentha longifolia (L.) Huds. Ml Leaf 5.73±0.28

Mentha x piperita L. Mp Leaf 6.80±0.19

Mentha pulegium L. Mpu Aerial 5.17±0.14

Micromeria croatica (Pers.) Schott Mc Aerial 7.90±0.24

Micromeria graeca (L.) Benth. ex Reich. Mg Aerial 9.92±0.07

Micromeria juliana (L.) Benth. ex Rchb. Mj Aerial 4.49±0.03

Micromeria thymifolia (Scop.) Fritsch Mt Aerial 7.67±0.22

Ocimum basilicum L. Ob Aerial 3.47±0.07

Origanum heracleoticum L. Oh Aerial 7.68±0.06

Origanum vulgare L. Ov Aerial 8.31±0.11

Rosmarinus officinalis L. Ro Leaf 5.27±0.11

Salvia officinalis L. So Leaf 3.46±0.11

Salvia sclarea L. Ss Leaf 4.95±0.09

Satureja montana L. Sm Aerial 5.51±0.05

Satureja subspicata Bartl. ex Vis. Ssu Aerial 11.03±0.04

Teucrium montanum L. Tm Aerial 8.20±0.24

Thymus longicaulis C. Presl Tl Aerial 5.48±0.17

Thymus pulegioides L. Tp Aerial 6.59±0.10

Thymus serpyllum L. subsp. serpyllum Ts Aerial 4.55±0.04

Thymus striatus Vahl Tst Aerial 3.13±0.05

Food Anal. Methods

while CA predominate in other 10 investigated species, twoslightly different analytical procedures, R-THA and C-THA,were used, and the total contents were expressed as RA andCA, respectively. Applied methods were based on the reac-tion of ortho-dihydroxyphenyl moiety of phenolic acids andnitrous acid forming yellow complexes, which turns orange-red in the presence of sodium hydroxide, and the absorbancewas read at 505 nm (RA) and 525 nm (CA), respectively.Flavonoids are polyphenolic compounds that also occur inthe medicinal plants of Lamiaceae family, but only some ofthem are o-dihydroxy phenols (for example, luteolin andquercetin) and may interfere with this quantification meth-od. However, taking into account their generally low contentin regard to phenolic acids (Blažeković et al. 2010;Nikolova 2011), their influence on the THA content mightbe considered negligible. It was found that the studiedLamiaceae plants differ greatly in their phenolic acids con-tents, ranging from 1.95 % (A. arvensis) to 11.03 %(Satureja subspicata). High amounts of phenolic acids werealso found in aerial parts of Micromeria graeca (9.92 %),leaves of M. officinalis (9.86 %), aerial parts of Origanumvulgare (8.31 %), Micromeria croatica (7.90 %), Origanumheracleoticum (7.68 %) and Micromeria thymifolia(7.67 %). Our results revealed that most of the studiedLamiaceae species originating from Croatia, especiallythose of the genus Micromeria, Origanum, Melissa, Menthaand Satureja, could be considered as a rich source ofhydroxycinnamic acids, mainly with the predominance ofRA.

Conclusions

In the present study, the prevalidation strategy to evaluateUV/Vis spectrophotometric method for THAs derivativesdetermination was carried out and successfully applied forthe quantitative analysis of 32 medicinal plants ofLamiaceae growing in Croatia. Since HPTLC analysis re-vealed that RA was predominant hydroxycinnamic acid inmost of the studied plants while in others CA preponderates,for quantification, two slightly different analytical proce-dures (R-THA and C-THA) were used. Prevalidation con-firmed the high quality of both investigated procedures withsomewhat higher sensitivity of R-THA procedure. Perfor-mance characteristics of both procedures showed ideal lin-ear calibration and ideal analytical evaluation functions,very low limits of quantitation, very high precision andaccuracy. Among the studied Lamiaceae species whichgreatly differ in their phenolic acids contents (1.95–11.03 %), our study highlighted especially those belongingto the genus Micromeria, Origanum, Melissa, Mentha andSatureja as a rich source of hydroxycinnamic acids, mainlywith the predominance of RA. The overall results

sufficiently demonstrate that both THA assays could beequally well applied for routine analysis of phenolic acidsin plant material as well as in searching for new rich naturalsources of these potent bioactive constituents.

Acknowledgement This research was funded by the Ministry ofScience, Education and Sports of the Republic of Croatia (Number ofthe project: 006-0061117-1238).

Conflict of Interest Maja Bival Štefan declares that she has noconflict of interest. Jadranka Vuković Rodríguez declares that she hasno conflict of interest. Biljana Blažeković declares that she has noconflict of interest. Marija Kindl declares that she has no conflict ofinterest. Sanda Vladimir-Knežević declares that she has no conflict ofinterest. This article does not contain any studies with human or animalsubjects.

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