Reagents and Materials
Azobisisobutyronitrile (AIBN, purum, ≥ 98%), ethylene glycol dimethacrylate (EGDMA, 99%), glycidyl methacrylate (GMA, ≥ 97%), L-histidine monohydrochloride monohydrate (HIS, ≥ 97%) and caffeine (≥ 99%) were purchased from Sigma-Aldrich (Buchs, Switzerland). Sodium carbonate (≥ 99%) was obtained from Carl Roth (Karlsruhe, Germany). Acetonitrile (ACN) and methanol (MeOH) were all HPLC grade (Chromasolv) and also from Sigma-Aldrich. Water was obtained from a Milli-Q water purification system from Merck Millipore (Billerica, Massachusetts).
Empty polypropylene SPE tubes (1 mL) with polyethylene frits (porosity 20 µm) were ordered from Sigma-Aldrich and Carl Roth. The SPE sorbent Oasis HLB (1 cm3, 30 mg) was acquired from Waters Corp. (Milford, Massachusetts, USA). Hydrophilized polytetrafluoroethylene (H-PTFE) filters were purchased from Machery Nagel (Düren, Germany).
Black and green tea samples were purchased from different manufacturers from Austria and Germany.
Instrumentation
All standard solutions, tea samples and SPE fractions were determined using a 1100 Series HPLC Value System from Agilent (Santa Clara, USA) equipped with a 1100 Series variable wavelength detector (VWD). Analyses were performed using an ACE C18-PFP (5 µm, 4.6 × 150 mm, Advanced Chromatography Technologies Ltd, Aberdeen, Scotland) analytical column. Mobile phase was a composition of 0.1% TFA in water (eluent A) and MeOH (eluent B). HPLC measurements were accomplished by a delayed gradient elution. The gradient program was performed using following steps (min/% eluent B): 0/5, 1/5, 28/63, 30/90, 32/90, 35/5, 40/5. The flow rate was 1 mL min− 1 and the injection volume was 20 µL. Temperature of the column oven was set to 40 °C and detection of tea samples and caffeine was performed at 280 nm.
Attenuated total-reflection infrared spectroscopy (ATR-IR) measurements of the polymers were executed on a Spectrum 100 FT-IR Spectrometer (Perkin Elmer, Waltham, USA) with a diamond measurement cell and a ZnSe crystal as focusing element. For each spectrum, ten scans were performed in the range between 4000 and 650 cm− 1 at a resolution of 4 cm− 1. For nitrogen sorption porosimetry a Quantachrome Nova Station 200 (Quantachrome Instruments, Boynton Beach, FL-USA) was employed. Adsorption studies were executed on a ThermoMixer C from Eppendorf (Hamburg, Germany). Separation of supernatants was performed by a Centrifuge 5418 from Eppendorf.
Preparation of Standard Solutions
Standard solution of caffeine (1000 µg mL− 1) was prepared in water. This stock solution was stored at 4 °C in the dark. Working standard solutions were daily prepared by diluting the stock solution with water.
Extraction and Analysis of Tea Samples
1 g of tea was extracted with 15 mL water in an ultrasonic bath at 70 °C for 15 min. After centrifugation the supernatant was collected. To ensure a complete extraction of caffeine, each sample was extracted for three times and the supernatants were combined in a 50 mL volumetric flask. Extracts were then diluted with water to 50 mL filtered using H-PTFE filters and transferred into glass vials for quantification by HPLC-UV.
Polymer Synthesis
Poly(GMA/EGDMA) particles were prepared by a thermally initiated polymerization procedure (Fig. 1). Briefly, 4.32 mL (33 mmol) GMA, 7.10 mL (38 mmol) EGDMA and 0.3 g AIBN (2 mmol) were dissolved in 45 mL ACN in a 100-mL round-bottomed flask. Then the solution was flushed with argon for 10 min to remove oxygen. Afterwards, the flask was equipped with a condenser, placed in an oil bath and polymerization was performed at 60 °C for 16 h under stirring. The resulting polymer was ground using mortar and pestle and thoroughly washed with water and MeOH. Finally, the polymer particles were dried at 50 °C.
Histidine Functionalization of Poly(GMA/EGDMA) Particles
For modification of poly(GMA/EGDMA), 1 g of sorbent was mixed with 25 mL 2 M sodium carbonate solution in a 100-mL round-bottomed flask equipped with a condenser followed by addition of 25 mL saturated l-histidine monohydrochloride monohydrate. Functionalization was then executed at 70 °C for 16 h under continuous stirring. The resulting polymer was finally washed with water and MeOH and dried at 45 °C under vacuum.
Adsorption Isotherms
Determination of the polymer capacity was accomplished by several batch adsorption experiments. Therefore 10–12 mg of polymer was placed into 2 mL reaction tubes followed by addition of 1 mL standard solution at concentrations ranging from 25 to 250 µg mL− 1. Then the tubes were placed in a thermo-mixer and incubated at room temperature for 1 h and 1200 rpm. Afterwards centrifugation was performed for 3 min at 14,000 rpm. Finally, the supernatants were filtered using H-PTFE filters and analyzed by HPLC-UV.
Adsorption Kinetics
Kinetic parameters of the adsorption process were determined in additional adsorption experiments. 10–12 mg of polymer was placed in 2 mL reaction tubes and 1 mL of a 100 mg L− 1 caffeine standard was added. Adsorption was then performed using a thermo-mixer at room temperature and 1200 rpm. Reactions were stopped at different time intervals ranging from 5 to 90 min. After centrifugation for 3 min at 14,000 rpm the supernatants were filtered using H-PTFE filters and analyzed by HPLC-UV.
Clean-up of Tea Samples/SPE Procedures
Synthesized polymers were tested for isolation of caffeine from different tea samples. First, 30 mg of sorbent was packed into 1 mL SPE cartridges with polyethylene frits above and below the sorbent bed. For conditioning 1 mL MeOH was applied to the cartridge followed by loading 0.5 mL of diluted tea extract 1:19 (v/v). After that elution of caffeine was executed using 3 mL of Milli-Q water. Solutions were finally quantified by HPLC-UV. Clean-up efficiency was further compared to the commercial available sorbent Oasis HLB. The Generic SPE protocol was obtained from Waters Corp [20]. Conditioning of the sorbent was executed using 1 mL MeOH followed by 1 mL Milli-Q water. Then 0.5 mL of diluted tea extract 1:19 (v/v) was applied to the polymer and subsequently washed with 1 mL 5% MeOH in water (v/v). Finally, elution was performed with 1 mL MeOH. Eluted fractions were evaporated to dryness, redissolved in 0.5 mL Milli-Q water and transferred to HPLC-UV analysis. All experiments were carried out in triplicate.
Method Validation
Performance evaluation of the HPLC method developed for determination of caffeine was accomplished according to international guidelines [21, 22]. The following parameters were determined: linearity, repeatability, accuracy, instrumental limits, method precision and stability of analytes.
Linearity was examined by measuring a standard solution (see “Preparation of Standard Solutions”) at concentrations between 25 and 200% of target concentration (20 µg mL− 1). For determination of the repeatability, a standard solution containing the target concentration level was prepared. Ten replicates per day were measured and repeated on three consecutive days to examine the intra-day and inter-day repeatability. Accuracy was investigated by spiking tea extracts with caffeine standard solutions at three different concentrations. Limit of detection (LOD) and limit of quantitation (LOQ) were determined from a calibration curve at concentrations ranging from 0.01 to 0.1 µg mL− 1. Calculations of instrumental limits were executed referring to DIN32645: 2008-11 (Eqs. 1 and 2).
$${\text{LOD}}=~\frac{{{s_{y.x}}}}{b} \cdot {t_{1 - \alpha ,v}} \cdot \sqrt {\frac{1}{N}+\frac{1}{n}+\frac{{{{\bar {x}}^2}}}{{\mathop \sum \nolimits_{{i=1}}^{N} {{\left( {{x_i} - \bar {x}} \right)}^2}}}}$$
(1)
$${\text{LOQ}}=k \cdot \frac{{{s_{y.x}}}}{b} \cdot {t_{1 - \alpha ,v}} \cdot \sqrt {\frac{1}{N}+\frac{1}{n}+\frac{{{{\left( {{\text{LOQ}} - \bar {x}} \right)}^2}}}{{\mathop \sum \nolimits_{{i=1}}^{N} {{\left( {{x_i} - \bar {x}} \right)}^2}}}}$$
(2)
\({s_{y.x}}\) is the residual standard deviation of y around the regression line, \(b\) is the slope, \(t_{1 - \alpha ,v}\) presents the student factor for 95% one sided for LOD and two sided for LOQ, \(N\) is the number of calibration points and \(n\) being the number of repetitions. Furthermore, the squared mean of x-values \({\bar {x}^2}\) and \(\mathop \sum \nolimits_{{i=1}}^{N} {\left( {{x_i} - \bar {x}} \right)^2}\) were calculated. The LOQ was iteratively determined. As recommended by DIN32645: 2008-11, \(k\) was set to 3. Convergences of LOQ quickly occurred and the result was taken when the percental change from one iteration step to the next was no more significant [23].
Degradation of employed analytes in standard solution was studied over 14 consecutive days. Therefore, the standard solution was stored at 4 °C in the dark and the amount of analyte was determined daily.