MS Method for the Quantification ... - ACS Publications

Subscriber access provided by UNIV OF WESTERN ONTARIO

Article

A validated LC-MS/MS method for quantification of free and bound lignans in cereal based diets and feces Natalja P. Nørskov, and Knud Erik Bach Knudsen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03452 • Publication Date (Web): 14 Oct 2016 Downloaded from http://pubs.acs.org on October 27, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 30

Journal of Agricultural and Food Chemistry

A validated LC-MS/MS method for quantification of free and bound lignans in cereal-based diets and feces Natalja P. Nørskov and Knud Erik Bach Knudsen

Aarhus University, Department of Animal Science, AU-Foulum, Blichers Alle 20, P.O. box 50 DK8830 Tjele, Denmark Corresponding author: Phone: +4587157724 Fax: +4587154249 E-mail: [email protected]

1 ACS Paragon Plus Environment


Page 2 of 30

1

Abstract

2

Despite the extensive literature describing the biological effects of phenolic compounds from

3

cereals, little is known about their bioaccessibility in the food matrix. This paper describes a

4

validated LC-MS/MS method for quantification of free and total content (free + bound) of eight

5

plant lignans (matairesinol, hydroxymatairesinol, secoisolariciresinol, lariciresinol, isolariciresinol,

6

syringaresinol, medioresinol and pinoresinol) and the two enterolignans (enterodiol and

7

enterolactone) in cereal based diets/bread and feces. The method consisted of alkaline methanolic

8

extraction combined with enzymatic hydrolyses, when measuring the total concentration of

9

lignans and methanolic extraction combined with enzymatic hydrolysis, when measuring free

10

lignans, followed by Solid Phase Extraction. The strength of this LC-MS/MS method is that it can be

11

combined with different types of samples, since the Solid Phase Extraction and LC-MS/MS

12

platform are similar to our previously published method for plasma and urine.

13 14 15

Keywords: plant lignans, enterolignans, LC-MS/MS, cereal diets, feces, bioaccessibility


Page 3 of 30


16

Introduction

17

The lignans form an interesting group of plant phenols which has a number of biological effects

18

such as antioxidant, antiviral, antibacterial, insecticidal, fungistatic, antitumor and

19

estrogenic/antiestrogenic effects1-9. In the Nordic countries, whole-grain cereals, in particular rye

20

and wheat, along with fruits and vegetables are the main source of plant lignans. Most of the

21

lignans in cereals are located in the bran, which constitutes the outermost part of the grain. They

22

are mainly bound to plant cell wall macromolecules, but a proportion of the lignans is free (not

23

bound to the cell wall), nevertheless they can be conjugated with one or two carbohydrate

24

moieties10,11. When ingested, lignans have to be hydrolyzed by the enzymes and bacteria in the

25

gastrointestinal tract to lignan aglycones, which can then be either absorbed directly or converted

26

by colonic bacteria to enterolignans, enterolactone and enterodiol12,13. The hydrolysis of lignans is

27

therefore an essential step for the bioaccessibility of plant lignans in the food matrix and further

28

their bioavailability in the body. The health effects of lignans depend on both the amount

29

consumed but also to a greater extent on their bioaccessibility in the food matrix14. To improve

30

the bioaccessibility in the food matrix, enzymatic treatment of the bran prior to consumption can

31

be performed. Studies on the enzymatic treatment of bran have shown positive results with

32

regard to yield of bioactive compounds14,15. Therefore there is a need to develop methods to

33

quantify both the total concentration of lignans (bound + free fraction) and the

34

released/bioaccessible lignans (free fraction).

35

The first quantitative method for lignans in food was published by Mazur et al 16 using GC-MS, in

36

which only two lignans, secoisolariciresinol and matairesinol were quantified. Two years earlier

37

Adlercreutz et al 17 published a GC-MS method for fecal samples in which three lignans were



Page 4 of 30

38

quantified, matairesinol and two enterolignans, enterodiol and enterolactone. Since then

39

extensive work has been done to quantify lignans in different types of food using different

40

extraction procedures and instruments. Milder et al 18 have published an LC-MS/MS method on

41

quantification of four plant lignans, secoisolariciresinol, matairesinol, lariciresinol and pinoresinol.

42

Penalvo et al 19 have published a GC-MS method in which the number of plant lignans was

43

extended to six. Smeds et al 20 succeeded in quantifying twenty-four lignans in sixteen different

44

cereal species using LC-MS/MS. The quantification of lignans using GC with electron capture

45

detector and LC with a coulometric electrode array detector has also been developed11,21.

46

Oppositely, only few methods have been developed to quantify lignans in feces. Heinonen et al 22

47

measured eight plant lignans and two enterolignans using HPLC with a coulometric electrode array

48

detector. To our knowledge no LC-MS/MS method on quantification of lignans in feces has been

49

published before.

50

We have previously developed a sensitive LC-MS/MS method to quantify eight plant lignans and

51

two enterolignans in plasma and urine23. Our purpose was therefore to extend the existing LC-

52

MS/MS method to be able to measure the lignans also in cereal-based diets/bread and in feces.

53

The use of a similar sample cleanup and LC-MS/MS platform can be a more simple approach to

54

measure lignans in different biological samples, compared to adapting different approaches and

55

instrumentation. Moreover, we have extended the extraction procedure to quantify not only the

56

total concentration of lignans (bound to the cell wall + free fraction), but also easily bioaccessible

57

lignans (free fraction, not bound to the cell wall) in both cereal and fecal samples. The methods

58

published so far on quantification of lignans in food focus on the total concentration of lignans.

59

We have developed a method to quantify the free fraction of lignans which can, for example, be of

60

interest in cases when the food matrix is enzymatically treated or treated by other means to 4 ACS Paragon Plus Environment

Page 5 of 30


61

increase the bioaccessibility of lignans. As with the cereal samples the lignans in fecal samples

62

were quantified as bound and free lignans, which to our knowledge has not been performed

63

before.

64

Material and methods

65

Material

66

The following standards were used: enterolactone, enterodiol, mataresinol, hydroxymataresinol,

67

secoisolariciresinol, lariciresinol, isolariciresinol, syringaresinol, medioresinol and pinoresinol from

68

Plantech (Berksher, UK) with purities of 96% and 95%. Glycocholic acid (Glycine-1 13C) from Sigma-

69

Aldrich (St. Louis, MO, USA) was used as an internal standard (IS). For the enzymatic hydrolysis β-

70

glucuronidase with an activity of ≥300000 units/g solid and a sulphatase activity of ≥10000 units/g

71

solid type H-1 from Helix pomatia was purchased from Sigma-Aldrich (St. Louis, MO, USA). Sodium

72

acetate and sodium hydroxide were obtained from Merck (Barmstadt, Germany). Glacial acetic

73

acid, formic acid, acetonitrile and methanol were purchased from Fluka/Sigma-Aldrich (St. Louis,

74

MO, USA). Acetonitrile and methanol were of HPLC grade.

75

Cereal samples. The cereal samples, used for the development and validation of the method, were

76

diets which were used in an ongoing pig study at Aarhus University. The diets consisted of refined

77

wheat supplemented with purified wheat fiber (Vitacel WF600, J. Rettenmeier and Söhne GmbH,

78

Rosenberg, Germany), rye bran and enzymatically treated rye bran. The enzymatic treatment of

79

the rye bran was done as described by Nielsen et al 24. The diets had an iso-dietary fiber content of

80

approx. 15%. The details of the study and diet ingredients will be described elsewhere. The diets

81

were cold-pelleted in a feed production unit at Aarhus University and stored at -20 °C. For the LC-

82

MS/MS analyses the diets were freeze-dried and finely milled to pass an 0.5 mm screen.



Page 6 of 30

83

Fecal samples. Fecal samples, used for the development and validation of the method, were

84

collected from the pigs, which were fed diets as described above. The pigs were initially fed the

85

refined wheat diets low in plant lignans as a wash-out diet. After the wash-out period the pigs

86

were switched to the lignan-rich rye bran diet for 8 days and then switched back to the refined

87

wheat diet for 4 days. The details of the study will be described elsewhere. Feces was collected in

88

the morning, freeze-dried and ground to a homogeneous powder. The feces from one pig was

89

used for the development and validation of the method. Further, we have analyzed the fecal

90

samples after 4 days on both diets.

91

Standards and standard curves

92

All standards and working solutions were prepared according to Nørskov et al

93

curves were prepared for quantitation of plant lignans; a standard curve containing a mixture of

94

standards (matairesinol, hydroxymatairesinol, secoisolariciresinol, lariciresinol, isolariciresinol,

95

syringaresinol, medioresinol and pinoresinol) in the range of 0.0488 – 100 ng/mL and a standard

96

curve containing only syringaresinol in the range of 1.56 – 800 ng/mL. This standard curve was

97

used to quantify syringaresinol in the rye bran when the concentration of syringaresinol was

98

higher than 100 ng/mL. The IS was added to both standard curves in a final concentration of 20

99

ng/mL. The standard curves were plotted as the analyte/internal standard concentration ratio

100

against the analyte/internal standard peak area ratio as a linear regression curve with 1/x

101

weighting. The standard curves showed good linearity with regression coefficients not lower than

102

0.998. The limits of detection (LODs) were estimated for each lignan in cereal and fecal samples

103

and defined as S/N = 3. The limits of quantification (LOQs) were defined as 5 x LODs.

104

Pretreatment of cereal and fecal samples prior LC-MS/MS

23

. Two standard


Page 7 of 30


105

The detailed schematic representation of the pretreatment procedure is shown in Figure 1. Part of

106

the pretreatment procedure was adapted from Penalvo et al 19 and Milder et al 18, but the weight

107

of the sample was lowered to adjust to our LC-MS/MS method. Both cereal and fecal samples

108

went through the same pretreatment procedure when analyzing the total lignan concentration

109

(bound +free fraction). Using this procedure we measured the total concentration of the eight

110

plant lignans. The concentration of free lignans, in case of cereal samples, was measured using

111

enzymatic hydrolysis whereas in the case of fecal samples no enzymatic hydrolysis was performed.

112

Method 1: Free lignans. The mammalian and plant lignans were extracted from 25 mg of cereal

113

and fecal samples by addition of 0.5 mL of MeOH, vortexed for 10 min. and centrifuged for 15 min

114

at 4 ˚C at 20,800 x g. The supernatant was transferred to a new tube and evaporated using N2 at

115

60 ˚C. Afterwards the fecal samples were redissolved in 0.6 mL of water and added 0.5 mL of

116

water containing 0.4% formic acid, centrifuged and the samples were now ready for solid phase

117

extraction (SPE). The cereal samples were incubated with 0.6 mL of freshly dissolved β-

118

glucuronidase/sulphatase at 37 ˚C for 19 h. Afterwards the samples were added 0.5 mL of water

119

containing 0.4 % of formic acid and centrifuged for 15 min at 4 ˚C. The samples were ready for SPE.

120

Method 2: Total lignans (bound + free). The plant lignans were extracted from the matrix using

121

alkaline hydrolysis as suggested by Penalvo et al 19 and Milder et al 18. A volume of 0.5 mL of 0.3 M

122

NaOH in 70% MeOH was added to 20 mg of cereal sample and 12 mg of fecal sample and

123

incubated in a vortex incubator at 60 ˚C for 1 h. The samples were cooled down and the pH was

124

adjusted with 20 µL of glacial acetic acid to a pH of approximately 5. Further, the samples were

125

centrifuged and the supernatant was transferred to another tube and evaporated under N2 flow at

126

60 ˚C. The hydrolysis of lignan glycosides was carried out with 0.6 mL of freshly dissolved β-



Page 8 of 30

127

glucuronidase /sulphatase (2 mg/mL in 50 mM sodium acetate buffer, pH 5) and incubated in a

128

vortex incubator at 37 ˚C for 19 h. The hydrolysis was stopped by addition of 0.4 % of formic acid

129

and the samples were centrifuged for 15 min at 4 ˚C. The samples were then ready for SPE.

130

SPE cleanup of cereal and fecal samples

131

The samples were cleaned up using SPE C18-E columns (55 µm, 70 Å with 50 mg/ 1 mL) or SPE

132

C18-E 96-well plates (55 µm, 70 Å with 25 mg/ well) from Phenomenex (Torrance, CA, USA), as in

133

the previously described SPE method for plasma and urine23. This method was slightly modified

134

and adjusted to cereal and fecal samples. To the preconditioned SPE columns or plates, the

135

samples were loaded and allowed to bind to the C18 material. The columns or plates were then

136

washed with 2 x 0.5 mL 5% methanol and the vacuum was applied to dry the sorbent. The lignans

137

were eluted with 400 µL of 100% acetonitrile and again the vacuum was applied to dry the

138

sorbent. The eluted samples were then diluted to 25% acetonitrile with 1200 µL water containing

139

glycocholic acid (Glycine-1

140

sampler correction and matrix effects validation. The samples were spun down at 20 ˚C for 5 min

141

prior to LC-MS/MS measurements. When measuring enterolactone in the fecal samples (free

142

fraction) the samples were diluted 100 times.

143

LC-MS/MS equipment and method

144

The samples were measured according to the LC-MS/MS method developed by Nørskov et al

145

using microLC 200 series from Eksigent/AB Sciex (Redwood City, CA, USA) equipped with a pre-

146

filter and Ascentis® C18 column, 100 mm x 1.0 mm with 3.0 µm particle size from Sigma-Aldrich

147

(St. Louis, MO, USA) and QTrap 5500 mass spectrometer from AB Sciex (Framingham, MA, USA).

148

The sample injection volume was 5 µL.

13

C) as IS to a final concentration of 20 ng/mL. IS was used for auto-

23


Page 9 of 30


149

Method validation. Validation of the method was implemented from the guidelines of the U.S.

150

Food and Drug Administration (FDA) 25 and the European Medicines Agency of Science Medicines

151

Health26.

152

Recovery. Recovery experiments were performed by spiking the known amount of lignan

153

standards to cereal and fecal samples immediately after addition of the extraction solvent, Figure

154

1. We have used cereal and fecal samples with low concentrations of lignans such as a refined

155

wheat diet and fecal samples from the period when the pigs were fed the refined wheat diet.

156

Appropriate volumes of working solution (400 ng/mL) containing a mixture of lignan standards

157

were added to the samples at three levels; low (5 x LLOQ=0.5 µg/100 g dry basis), medium (25 x

158

LLOQ=15 µg/100 g dry basis) and high (ULOQ=250 µg/100 g dry basis).

159

Accuracy and Precision. Accuracy and precision (intra-batch) of the method were assured by the

160

addition of standards at three concentrations, low (0.195 ng/mL), medium (6.25 ng/mL) and high

161

(100 ng/mL) after the SPE cleanup of different samples. This would allow the validation of the

162

matrix effects and variation, which is essential when developing the LC-MS/MS method.

163

Moreover, in the case of cereals we have validated the two matrixes: refined wheat (simple

164

matrix) and rye bran (complex matrix). The inter-batch variation was calculated based on Quality

165

control (QC) samples of refined wheat, rye bran and fecal samples from the period when the pigs

166

were fed refined wheat, n=10. The accuracy, precision and matrix factor (MF) were calculated as

167

descried by Nørskov et al23 . Briefly, the accuracy was calculated as a relative error (ER) of

168

replicated measurements, n=5, whereas the precision was calculated as the relative standard

169

deviation (RSD) of replicated measurements n=5, with acceptance criteria of ±20% at the low

170

concentration and ±15% at the medium and high concentrations. We have used our previous



Page 10 of 30

171

approach to validate and calculate the matrix effects, where the MF for each lignan and IS were

172

calculated as the ratio between the peak area in the presence of the matrix and in the absence of

173

the matrix, which should be close to 100 %.

174

Results and discussion

175

Method validation.

176

Sample hydrolysis. To release the esterified lignans from the cereal or fecal matrix we have used

177

the combination of methanolic extraction with alkaline hydrolysis. The released lignan glycosides

178

were then enzymatically hydrolysed by the enzymes β-glucuronidase/sulfatase to lignan

179

aglycones and quantified as total lignans in the sample (Method 2). The idea of methanolic

180

extraction with alkaline hydrolysis and further enzymatic hydrolysis was adapted from Penalvo et

181

al 19 and Milder et al 18 and optimized with regard to weight of the sample to our LC-MS/MS

182

method. However, hydroxymataresinol has been reported to be unstable in alkaline hydrolysis,

183

forming conidendric acid and α-conidendrin among other compounds 20. We were therefore not

184

able to detect hydroxymatairesinol after alkaline hydrolysis (Method 2), which comfirmed the

185

previous findings by Smeds et al 20. In Method 1 only enzymatic hydrolysis was performed for

186

cereal samples to release the lignan aglycones, which provided the measure of the amount of the

187

free lignans not bound to the plant matrix. The fecal samples in Method 1 were not treated with

188

enzymes, because our preliminary results showed that no additional lignans were released when

189

using enzymes hydrolysis (data not shown). Heinonen et al 22 have also reported that < 10 % of the

190

enterolignans are conjugated in feces.


Page 11 of 30


191

Accuracy, precision and recovery. The analyses of lignans in cereals and also in feces can be a

192

challenging task because of their complex matrixes . Therefore we have used different approaches

193

to validate our LC-MS/MS method. To investigate the possible losses during different extraction

194

procedures we have added standards from the beginning of the sample pretreatment, Figure 1.

195

The results are summarized in Table 1. Standards were added to the cereal and fecal samples in

196

three concentrations, low, medium and high. The concentration of several lignans in both cereal

197

and fecal samples was too high to be able to calculate the recovery at the low concentration.

198

We did not observe any major losses of lignans during different extractions, but the recoveries for

199

Method 1 were generally better than for Method 2. This was also expected because Method 2

200

included more extraction steps compared to Method 1. In case of Method 2 the recoveries at the

201

low concentration varied from 83 to 95%, at the medium concentration from 78 to 108% and at

202

the high concentration from 71 to 99%, which is comparable to recoveries of plant lignans

203

reported by Penalvo et al 19 and Milder et al 18. The lowest recovery was measured for

204

matairesinol, which is in agreement with the observation of Penalvo et al 19 and Milder et al 18 that

205

matairesinol is probably slightly unstable during the alkaline hydrolysis. However, the recovery of

206

matairesinol was not that low compared to recoveries of 50% reported by Penalvo et al 19 and

207

Milder et al 18, and it was mainly affected at the high concentrations; 71% for fecal samples and

208

79% for cereal samples. At the low and medium concentrations the recovery of matairesinol was

209

higher and comparable to other plant lignans, Table 1. Because matairesinol has the lowest

210

abundancy in both cereal and fecal samples, it is measured at a low concentration and therefore

211

we decided to keep matairesinol in Method 2. The majority of lignans in Method 1 had recoveries

212

close to 100%. To measure free lignans as a separate fraction has, to our knowledge, not been

213

performed before and therefore the results can not be compared to other studies. 11 ACS Paragon Plus Environment


Page 12 of 30

214

To validate the influence of the matrix of cereal and fecal samples, the standards of three

215

concentrations (low, medium and high) were added to the samples in the presence and absence of

216

the matrix, and the accuracy and precision were calculated, Table 2. In general the accuracy and

217

precision (intra-batch) for all the lignans met the acceptance criterion that it should not deviate by

218

more than 15 %. Penalvo et al 19 have also reported intra-batch and inter-batch variation of < 14%

219

indicating that the method is repeatable. The inter-batch variation in our method was slithly

220

higher, 18 % compared to Penalvo et al 19. However, there were some differences in accuracy and

221

precision among the different lignans. The accuracy and precision were lowest for pinoresinol and

222

medioresinol with accuracies of 9 – 14%, whereas for other lignans it was higher, < 9%, Table 2. It

223

was also noticed that the accuracy and precision of the lignans were lower in the complex

224

matrixes such as rye bran and feces compared to refined wheat. Milder et al 18 have also

225

experienced high ion suppression by a complex matrix such as whole grain wheat bread. To get a

226

better measure of the matrix effects we used IS, 13C-labaled glycocholic acid, to calculate the MF.

227

Isotope-labeled glycocholic acid is much cheaper compared to the isotope-labeled lignan

228

standards and was proven to be influenced by the ion suppresson similar to the lignans in our

229

previous study 23. Our calculations on the MF for Method 1 were: 98% (refined wheat), 94% (rye

230

bran) and 93% (feces) indicating that no ion suppression had occurred when analysing the free

231

lignan fraction. Our calculation on the MF for Method 2 has shown much stronger matrix effects

232

with a signal decrease up to 35 % for rye bran and fecal samples. Because alkaline hydrolysis

233

released not only lignans but also many other compounds, the matrix was saturated with

234

compounds interfering during ionisation. However, in spite of the decrease in signal, IS

235

compensated for the loss when calculating lignan concentrations.


Page 13 of 30


236

Sensitivity and selectivity. The sensitivity and selectivity are the key parameters of the LC-MS/MS

237

method, which are also compound and instrument dependent. When using MRM mode the

238

selectivity is high allowing quantification at low concentrations. Nevertheless, background noice

239

and interfering peaks can influence the selectivity and sensitivity of each compound differently.

240

Figures 2 and 3 show the selectivity for each lignan in two different matrixes using the two

241

extraction methods, Methods 1 and 2. Figure 2 shows the selectivity of plant lignans at the LOQ

242

level in refined wheat as a matrix and Figure 3 shows the selectivity of plant lignans and

243

enterolignans at the LOQ level in feces. In both cases a higher background noice and more

244

interfering peaks appeared after extraction with alkaline hydrolysis, which can be explained by the

245

fact that alkaline hydrolysis promoted the break-down of matrix componets and the release of

246

many more matabolites. In cereal samples pinoresinol and medioresinol had a lower selectivity

247

compared to other plant lignans, which also comfirmed their lower accuracy as discussed in the

248

previous section. After extraction, the chromatograms from Method 1 had fewer additional peaks

249

indicating a cleaner matrix, which improved the selectivity. The sensitivity of the method was

250

estimated in three matrixes, refined wheat diet, rye bran diet and feces, and summarized in Table

251

3. The LODs for enterolignans in feces were low at around 0.05 µg/100 g dry basis for enterediol

252

and 0.08 µg/100 g dry basis for enterolactone. For plant lignans (matairesinol,

253

hydroxymatairesinol, secoisolariciresinol, lariciresinol and isolariciresinol) the LODs varied from

254

0.12 to 0.43 µg/100 g dry basis whereas the LODs were higher for syringaresinol, pinoresinol and

255

medioresinol varying from 2 to 10 µg/100 g dry basis, Table 3. The LODs for most of the lignans in

256

our method are better compared to previous quantification methods reported by Penalvo et al 19,

257

Milder et al 18 and Mazur et al 16. The quantification of lariciresinol, secoisolariciresinol ,

258

mataresinol and pinoresinol by Milder et al 18 was also performed by LC-MS/MS and therefore this



Page 14 of 30

259

method is comparable to our method. However, their LODs varied from 4 to 10 µg/100 g of DM

260

and therefore only pinoresinol is comparable. The improved LODs in our method can be ascribed

261

to a combination of the improved sensitivity of a newer MS instrument and a different cleanup

262

procedure used before the LC-MS/MS measurements. We performed an SPE cleanup of the

263

samples instead of the liquid liquid extraction (LLE) performed by Milder et al 18. An SPE cleanup of

264

the samples is known to give cleaner samples compared to LLE. However, Penalvo et al 19 also

265

reported higher LODs varying between 6 and 16 µg/100 g dry basis using an SPE cleanup combined

266

with GC-MS. Other studies have also reported higher LODs in solid food using HPLC-APCI-MS and

267

coulemetric electrode array detection 27,28.

268

Lignan content in cereal and fecal samples. The developed LC-MS/MS method was used to

269

quantify lignans in cereal-based diets, bread and feces and the results are summarized in Tables 4,

270

5, and 6 and based on duplicate determination. Eight different plant lignans were included in the

271

method representing the most abundant lignans in cereals known so far 19,20. In fact, syringaresinol

272

was the most abundant lignan in rye, thus confirming previous results of Penalvo et al 19 and

273

Smeds et al 20. As expected, Table 4, the concentration of lignans in the free fraction (Method 1)

274

was much lower compared to the total concentration of lignans (bound + free fraction, Method 2).

275

Nevertheless, the proportion of free relative to total lignans in rye bran was 35 % compared to

276

enzyme-treated rye bran where the proportion was 52 % indicating that more lignans (17 %) were

277

released from the matrix as a consequence of the enzyme treatment with cell wall degrading

278

enzymes. Bioprocessing of bran with enzymes has also been found to improve the bioaccessibility

279

of ferulic acid from 1.1 % to 5.5 % 14. In another study the concentration of total phenolic

280

compounds increased when rye bran was enzymatically treated 15. This is in concert with our

281

results that clearly showed that lignans are highly influenced by the enzymatic treatment, 14 ACS Paragon Plus Environment

Page 15 of 30


282

encreasing free lignan fraction Table 4. The concentration of lignans in the refined wheat diet was,

283

as expected, low due to the debranning of wheat.

284

The method has also been used to quantify lignans in bread with increasing amount of dietary

285

fiber (DF) provided as bran. Increased amounts of lignans correlated well with increased DF

286

content, Table 5, and there was also a good agreement with previously analysed rye bread by

287

Milder et al 18. The study on the content of lignans in cereal brans performed by Smeds et al 20 also

288

showed a high concentration of lignans in rye. However, since the measurements were performed

289

on dried grains and not on bread, the results are not directly comparable with our study. The

290

results of Smeds et al 20 also showed that the yield of lignans is influenced by the extraction used,

291

and that ASE gives the higher yield of syringaresinol, pinoresinol and hydroxymatairesinol

292

compared to alkaline extraction. However, ASE requires instrumentation, and therefore other

293

alternatives such as alkaline extration or alkaline extraction combined with acid extraction can be

294

applied. We have chosen the combination of alkaline and enzymatic hydrolysis since it provided

295

reproducable results and was succesfully applied by Milder et al 18 and Penalvo et al 19.

296

The concentration of lignans in feces was low when the pigs were fed the refined wheat diet (after

297

4 days) and high when the pigs were fed the rye bran diet (after 4 days), Table 6. This is in

298

agreement with our previous pig studies on lignans in urine and plasma 23,29,30. The free fraction of

299

lignans in feces was primarily present as enterolignans, enterolactone and enterodiol whereas

300

only a very small proportion of plant lignans occurred as free. In the bound + free lignan fraction,

301

besides the enterolactone, the concentration of syringaresinol was high followed by lariciresinol

302

indicating that a high proportion of syringaresinol was not released in the gastrointestinal tract

303

and was excreted in feces bound to the plant matrix. This is also the case for lariciresinol and



Page 16 of 30

304

isolariciresinol, which were not detected in the free fraction either, but only as bound lignans.

305

Oppositely, enterolactone and enterodiol were quantified in the free fraction and no additional

306

signal was detected in the bound fraction (data not shown). Our results are in good agreement

307

with a previous study with pigs in which the concentration of enterolactone in feces after one

308

week on a rye diet was 70.9 µmol/kg dry matter and enterodiol 2.1 µmol/kg dry matter 30. In the

309

present study we measured the concentration of enterolactone after 7 days on the rye bran diet

310

to 61 µmol/kg dry matter and enterodiol to 0.1 µmol/kg dry matter.

311

The strength of the developed LC-MS/MS method is that it can be used for a high number of cereal

312

and fecal samples and can also be combined with plasma and urine samples 23. The similar SPE

313

cleanup, chromatography and MRM method for different biological samples are easier to adapt

314

compared to different instrumentation for each type of sample.


Page 17 of 30


References 1. Willfor, S. M.; Ahotupa, M. O.; Hemming, J. E.; Reunanen, M. H. T.; Eklund, P. C.; Sjoholm, R. E.; Eckerman, C. S. E.; Pohjamo, S. P.; Holmbom, M. R. Antioxidant activity of knotwood extractives and phenolic compounds of selected tree species. J. Agric. Food. Chem. 2003, 51, 7600-7606. 2. Saarinen, N. M.; Warri, A.; Makela, S. I.; Eckerman, C.; Reunanen, M.; Ahotupa, M.; Salmi, S. M.; Franke, A. A.; Kangas, L.; Santti, R. Hydroxymatairesinol, a novel enterolactone precursor with antitumor properties from coniferous tree (Picea abies). Nutrition and Cancer-an International Journal 2000, 36, 207-216. 3. Kitts, D. D.; Yuan, Y. V.; Wijewickreme, A. N.; Thompson, L. U. Antioxidant activity of the flaxseed lignan secoisolariciresinol diglycoside and its mammalian lignan metabolites enterodiol and enterolactone. Mol. Cell. Biochem. 1999, 202, 91-100. 4. Thompson, L. U.; Seidl, M. M.; Rickard, S. E.; Orcheson, L. J.; Fong, H. H. S. Antitumorigenic effect of a mammalian lignan precursor from flaxseed. Nutrition and Cancer-an International Journal 1996, 26, 159-165. 5. Thompson, L. U.; Rickard, S. E.; Orcheson, L. J.; Seidl, M. M. Flaxseed and its lignan and oil components reduce mammary tumor growth at a late stage of carcinogenesis. Carcinogenesis 1996, 17, 1373-1376. 6. Saarinen, N. M.; Huovinen, R.; Warri, A.; Makela, S. I.; Valentin-Blasini, L.; Needham, L.; Eckerman, C.; Collan, Y. U.; Santti, R. Uptake and metabolism of hydroxymatairesinol in relation to its anticarcinogenicity in DMBA-induced rat mammary carcinoma model. Nutrition and Cancer-an International Journal 2001, 41, 82-90. 7. Saarinen, N. M.; Smeds, A.; Makela, S. I.; Ammala, J.; Hakala, K.; Pihlava, J. M.; Ryhanen, E. L.; Sjoholm, R.; Santti, R. Structural determinants of plant lignans for the formation of enterolactone in vivo. J. Chromatogr. B-Analyt. Technol. Biomed. Life Sci. 2002, 777, 311-319. 8. Oikarinen, S. I.; Pajari, A.-M.; Mutanen, M. Chemopreventive activity of crude hydroxsymatairesinol (HMR) extract in ApcMin mice. Cancer Letters 2000, 161, 253-258. 9. Katsuda, S.; Yoshida, M.; Saarinen, N.; Smeds, A.; Nakae, D.; Santti, R.; Maekawa, A. Chemopreventive effects of hydroxymatairesinol on uterine carcinogenesis in Donryu rats. Experimental Biology and Medicine 2004, 229, 417-424. 10. Bondia-Pons, I.; Aura, A. M.; Vuorela, S.; Kolehmainen, M.; Mykkanen, H.; Poutanen, K. Rye phenolics in nutrition and health. Journal of Cereal Science 2009, 49, 323-336. 11. Schwartz, H.; Sontag, G. Determination of secoisolariciresinol, lariciresinol and isolariciresinol in plant foods by high performance liquid chromatography coupled with coulometric electrode array detection. J. Chromatogr. B-Analyt. Technol. Biomed. Life Sci. 2006, 838, 78-85.



Page 18 of 30

12. Peterson, J.; Dwyer, J.; Adlercreutz, H.; Scalbert, A.; Jacques, P.; McCullough, M. L. Dietary lignans: physiology and potential for cardiovascular disease risk reduction. Nut. Rev. 2010, 68, 571603. 13. Bolvig, A. K.; Adlercreutz, H.; Theil, P. K.; Jorgensen, H.; Knudsen, K. E. B. Absorption of plant lignans from cereals in an experimental pig model. Br. J. Nut. 2016, 115, 1711-1720. 14. Anson, N. M.; Selinheimo, E.; Havenaar, R.; Aura, A. M.; Mattila, I.; Lehtinen, P.; Bast, A.; Poutanen, K.; Haenen, G. R. M. M. Bioprocessing of Wheat Bran Improves in vitro Bioaccessibility and Colonic Metabolism of Phenolic Compounds. J. Agric. Food. Chem. 2009, 57, 6148-6155. 15. Radenkovs, V.; Klava, D.; Krasnova, I.; Juhnevica-Radenkova, K. Application of enzymatic treatment to improve the concentration of bioactive compounds and antioxidant potential of wheat and rye bran. In 9th Baltic Conference on Food Science and Technology - Food for Consumer Well-Being: Foodbalt 2014, Straumite, E., Ed. 2014; pp 127-132. 16. Mazur, W.; Fotsis, T.; Wahala, K.; Ojala, S.; Salakka, A.; Adlercreutz, H. Isotope dilution gas chromatographic mass spectrometric method for the determination of isoflavonoids, coumestrol, and lignans in food samples. Anal. Biochem. 1996, 233, 169-180. 17. Adlercreutz, H.; Fotsis, T.; Kurzer, M. S.; Wahala, K.; Makela, T.; Hase, T. Isotope-Dilution GasChromatographic Mass-Spectrometric method for the determination of unconjugated lignans and isoflavonoids in human feces, with preliminary-results in omnivorous and vegetarian women. Anal. Biochem. 1995, 228, 358-358. 18. Milder, I. E. J.; Arts, L. C. W.; Venema, D. P.; Lasaroms, J. J. P.; Wahala, K.; Hollman, P. C. H. Optimization of a liquid chromatography-tandem mass spectrometry method for quantification of the plant lignans secoisolariciresinol, matairesinol, lariciresinol, and pinoresinol in foods. J. Agric. Food. Chem. 2004, 52, 4643-4651. 19. Penalvo, J. L.; Haajanen, K. M.; Botting, N.; Adlercreutz, H. Quantification of lignans in food using isotope dilution gas chromatography/mass spectrometry. J. Agric. Food. Chem. 2005, 53, 9342-9347. 20. Smeds, A. I.; Eklund, P. C.; Sjoholm, R. E.; Willfor, S. M.; Nishibe, S.; Deyama, T.; Holmbom, B. R. Quantification of a broad spectrum of lignans in cereals, oilseeds, and nuts. J. Agric. Food. Chem. 2007, 55, 1337-1346. 21. Cukelj, N.; Jakasa, I.; Sarajlija, H.; Novotni, D.; Curic, D. Identification and quantification of lignans in wheat bran by gas chromatography-electron capture detection. Talanta 2011, 84, 127132. 22. Heinonen, S.; Nurmi, T.; Liukkonen, K.; Poutanen, K.; Wahala, K.; Deyama, T.; Nishibe, S.; Adlercreutz, H. In vitro metabolism of plant lignans: New precursors of mammalian lignans enterolactone and enterodiol. J. Agric. Food. Chem. 2001, 49, 3178-3186.


Page 19 of 30


23. Norskov, N. P.; Olsen, A.; Tjonneland, A.; Bolvig, A. K.; Laerke, H. N.; Knudsen, K. E. B. Targeted LC-MS/MS Method for the Quantitation of Plant Lignans and Enterolignans in Biofluids from Humans and Pigs. J. Agric. Food. Chem. 2015, 63, 6283-6292. 24. Nielsen, T. S.; Jensen, B. B.; Purup, S.; Jackson, S.; Saarinen, M.; Lyra, A.; Sorensen, J. F.; Theil, P. K.; Knudsen, K. E. B. A search for synbiotics: effects of enzymatically modified arabinoxylan and Butyrivibrio fibrisolvens on short-chain fatty acids in the cecum content and plasma of rats. Food & Function 2015, 7, 1839-1848. 25. Guidance for Industry: Bioanalytical Mthod Validation. U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER), Center of Veterinary Medicine (CVM), May 2001. Available on the internet at http://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/default.htm. 26. Guideline on bioanalytical method validation. Eurpean Medicines Agency of Science Medicine Health. Committee for Medicinal Products for Human Use (CHMP), February 2012. Available on the internet at http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2011/08/WC500 109686.pdf. 27. Horn-Ross, P. L.; Barnes, S.; Lee, M.; Coward, L.; Mandel, J. E.; Koo, J.; John, E. M.; Smith, M. Assessing phytoestrogen exposure in epidemiologic studies: development of a database (United States). Cancer Causes & Control 2000, 11, 289-298. 28. Kraushofer, T.; Sontag, G. Determination of matairesinol in flax seed by HPLC with coulometric electrode array detection. J. Chromatogr. B-Analyt. Technol. Biomed. Life Sci. 2002, 777, 61-66. 29. Laerke, H. N.; Mortensen, M. A.; Hedemann, M. S.; Knudsen, K. E., Bach; Penalvo, J. L.; Adlercreutz, H. Quantitative aspects of the metabolism of lignans in pigs fed fibre-enriched rye and wheat bread. Br. J. Nut. 2009, 102, 985-994. 30. Knudsen, K. E. B.; Serena, A.; Kjaer, A. K. B.; Tetens, I.; Heinonen, S. M.; Nurmi, T.; Adlercreutz, H. Rye bread in the diet of pigs enhances the formation of enterolactone and increases its levels in plasma, urine and feces. J. Nut. 2003, 133, 1368-1375.



Page 20 of 30

Figure captions Figure 1. Pretreatment of samples before LC-MS/MS quantification. Figure 2. Selectivity of Method 1 (A) and Method 2 (B) in cereal samples spiked with a low concentration standard (0.195 ng/mL) except pinoresinol and medioresinol (6.25 ng/mL). Figure 3. Selectivity of Method 1 (A) and Method 2 (B) in fecal samples spiked with a low concentration standard (0.195 ng/mL) except syringaresinol, pinoresinol and medioresinol (6.25 ng/mL).


Page 21 of 30


Table 1. Recovery % of Lignans, Extraction Method 1 (Free Lignans) and Method 2 (Total Lignans) in Cereal and Fecal samples at Three Levels, Low (L) = 0.5 µg/100 g dry basis, Medium (M) = 15 µg/100 g dry basis and High (H) = 250 µg/100 g dry basis, (n=5). Cereal samples Method 1

Fecal samples

Method 2

Method 1

enterolactone

L -

M -

H -

L -

M -

H -

L a nq

Enterodiol

-

-

-

-

-

-

nq

matairesinol

98 ±7 nq

108 ±3 102 ±4 103 ±2 95 ±3 107 ±4 93 ±6 99 ±6 90 ±3

95 ±5 ndb

82 ±3 nd

79 ±2 nd

83 ±7 nq

83 ±5 96 ±5 84 ±6 88 ±6 nq

85 ±2 94 ±2 85 ±1 86 ±1 82 ±3 87 ±2

106 ±14 104 ±10 86 ±8 108 ±15 105 ±8 nd

hydroxymataresinol secoisolariciresinol lariciresinol

97 ±4 nq

pinoresinol

101 ±2 nd

syringaresinol

nd

medioresinol

nd

isolariciresinol

a b

109 ±4 106 ±2 99 ±2 96 ±3 109 ±1 103 ±4 98 ±7 104 ±3

94 ±5 nd nd nd

94 ±6

nd nd

M 97 ±12 97 ±3 108 ±4 90 ±5 100 ±4 94 ±3 103 ±2 106 ±5 106 ±7 92 ±5

H 92 ±3 94 ±3 102 ±2 88 ±3 93 ±4 94 ±3 99 ±3 105 ±2 106 ±4 103 ±7

Method 2 L nq nq nq nd nq

M 108 ±17 95 ±3 83 ±5 nd

nd

78 ±3 91 ±8 93 ±5 100 ±11 nq

nd

nq

nq nq nd

H 99 ±2 99 ±3 71 ±3 nd 74 ±5 84 ±4 81 ±3 82 ±4 94 ±5 84 ±4

Not quantified Not detected

21

ACS Paragon Plus Environment


Page 22 of 30

Table 2. Accuracy and Precision (Intra-Batch) of Lignans, Extraction Method 1 (Free Lignans) and Method 2 (Total Lignans) in Cereal and Fecal Samples at Three Levels, Low (L)=0.195 ng/mL, Medium (M)=6.25 ng/mL and High (H)=100 ng/mL, Shown as a Range (minmax) of Eight Lignans in Case of Cereal Samples and Ten in Case of Fecal Samples. Method 1 Precision intra-batch (±RSD %)

Accuracy (RE %)

cereal samples (refined wheat) cereal samples (rye bran) fecal samples a Not quantified

L 5-7

M 0.2-7

H 1-8

L 5-12

M 1-6

nqa

0.2-11

2-10

nq

2-6

5-12

2-11

0.7-9

4-15

2-7

H 0.2-0.7

Method 2 Accuracy (RE %)

Precision intra-batch (±RSD %)

L 7-11

M 5-11

H 2-10

1-5

nq

2-13

2-14

0.9-3

nq

4-14

2-14

L 4-14

M 1-7

H 1-3

nq

2-7

2-4

nq

2-14

1-3

22

ACS Paragon Plus Environment

Page 23 of 30


Table 3. Limits of Detection (LODs) and Limits of Quantification (LOQs) for Lignans in Cereals and Feces, Dry Basis (using Extraction Method 1). a

LODs µg/100 g enterolactone enterodiol matairesinol hydroxymataresinol secoisolariciresinol lariciresinol isolariciresinol pinoresinol syringaresinol medioresinol

c

0.08 c 0.05 0.12 0.15 0.31 0.43 0.35 2-5d d 2-5 d 7-10

b

LOQs µg/100 g c

0.40 c 0.25 0.61 0.75 1.6 2.2 1.8 10-25d d 10-25 d 35-50

a

Limits of detection estimated in cereals and feces, S/N=3 Limits of quantification estimated in cereals and feces, 5 x LOD c Only fecal samples d Depends on the matrix b



Page 24 of 30

Table 4. Content of Lignans (µg/100 g Dry Matter) in Cereal-Based Diets, Extraction Method 1 (Free Lignans) and Method 2 (Total Lignans), n=2. Refined wheat matairesinol hydroxymataresinol secoisolariciresinol lariciresinol isolariciresinol pinoresinol syringaresinol medioresinol total a

Rye bran

Method 1

Method 2

Method 1

Method 2

0.9 ±0.1 8 ±0.1 1 ±0 3 ±0.1 0.6 ±0.1 nd 19 ±1 4 ±0.3 27 ±3

0.7 ±0.1 a nd 2 ±1 11 ±0.6 2 ±0.1 8 ±0.8 77 ±12 40 ±1 141 ±14

7 ±0.4 9 ±0.1 5 ±0.5 64 ±6 6 ±0.1 6 ±0.5 590 ±23 26 ±6 703 ±24

20 ±1 nd 20 ±1 172 ±11 30 ±2 47 ±3 1598 ±95 131 ±16 2017 ±98

Enzymatically treated rye bran Method 1 9 ±0.2 9 ±0.1 6 ±0.3 50 ±0.1 21 ±0.3 5 ±1 1247 ±5 39 ±11 1376 ±9

Method 2 18 ±3 nd 24 ±3 132 ±17 36 ±5 35 ±8 2393 ±100 142 ±11 2781 ±146

Not detected


Page 25 of 30


Table 5. Content of Lignans (µg/100 g Dry Matter) Extraction Method 2 (Total Lignans) in Rye Bread with increasing Content of Dietary Fiber, n=2. Bread 1 DFa 150 g/kg dry matter matairesinol hydroxymataresinol secoisolariciresinol lariciresinol isolariciresinol pinoresinol syringaresinol medioresinol total a b

18 ±1 b nd 25 ±1 229 ±7 30 ±0.1 92 ±3 1459 ±23 294 ±15 2147 ±11

Bread 2 DF 189 g/kg dry matter 23 ±1 nd 30 ±1 263 ±3 35 ±4 139 ±3 1433 ±52 360 ±21 2283 ±98



Dietary fiber Not detected



Page 26 of 30

Table 6. Content of Lignans (µg/100 g Dry Matter) in Fecal Samples, Extraction Method 1 (Free Lignans) and Method 2 (Total Lignans), n=2. The Pigs were fed a Rye Bran Diet and a Refined Wheat Diet, both Diets for 4 Days. Refined wheat enterolactone enterodiol Total enterolignans matairesinol hydroxymataresinol secoisolariciresinol lariciresinol isolariciresinol pinoresinol syringaresinol medioresinol Total plant lignans a b

Method 1

Method 2

71 ±1 2 ±0.1 73 ±1 0.7 ±0.1 0.3 ±0.1 1 ±0.5 b nd nd nd 6 ±2 nd 7 ±3

7 ±0.5 nd 15 ±0.2 44 ±7 7 ±0.0 3 ±4 423 ±20 nd 498 ±23

a

Rye bran Method 1

Method 2

2615 ±56 18 ±0.3 2632 ±57 7 ±0.2 0.5 ±0.3 4 ±0.2 nd nd 6 ±3 28 ±4 nd 45 ±1

49 ±1 nd 26 ±3 164 ±11 44 ±9 78 ±8 1312 ±23 nd 1673 ±27

Enterolactone and enterodiol were quantified using Method 1 Not detected


Page 27 of 30


Method 1: Free lignans

Method 2: Total lignans (bound + free)

25 mg of cereal or fecal sample (2 mL tube)

20 mg of cereal 12 mg of fecal sample (2 mL tube)

Addition of 0.5 mL MeOH

Addition 0.5 mL of 0.3 M NaOH in 70 % MeOH

Vortex for 10 min.

Vortex for 2 min. and incubation (vortex incubator) for 1 h 60 ˚C Neutralisation with 20 µL of acetic acid

Addition of standards for recovery experiments

Vortex for 1 min. Centrifugation for 15 min. 4 ˚C 20817 x g Supernatant collection in a new tube (2 mL tube) Supernatant evaporation to dryness by N2 flow 60 ˚C Fecal samples (free lignans): addition of 0.6 mL of water

Cereal samples (free and total lignans) and fecal samples (total lignans): addition of 0.6 mL of enzymes

Vortex for 5 min. Incubation (vortex incubater) 37 ˚C for 19 h. Addition of 0.5 mL 0.4 % Formic acid Centrifugation for 15 min. 4 ˚C 20817 x g SPE cleanup Addition of standards for accuracy experiments

LC-MS/MS

Figure 1.



Page 28 of 30

A

B

Figure 2.


Page 29 of 30


A

B

Figure 3.



Page 30 of 30

TOC


MS Method for the Quantification ... - ACS Publications

Recommend Documents