Shuguang Li, Jason Anspach, Sky Countryman, and Erica Pike Phenomenex, Inc., 411 Madrid Ave., Torrance, CA USA PO _W

Transcription

1 Simple, Fast and Accurate Quantitation of Human Plasma Vitamins and Their Metabolites by Protein Precipitation Combined with Columns Using HPLC-UV, HPLC-FLD or LC/MS/MS Shuguang Li, Jason Anspach, Sky Countryman, and Erica Pike Phenomenex, Inc., 411 Madrid Ave., Torrance, CA USA PO _W

2 Abstract Monitoring the body s concentration of vitamins and their metabolites (e.g. phosphates) is an aid in diagnosing and treating disease states related to their deficiencies. The number of laboratories that analyze vitamins and their metabolites has risen dramatically, a majority of which are under pressure to rapidly and accurately process patient samples. It is important that the analytical method be rapid, sensitive, and accurate in order to accommodate these high-throughput labs that analyze thousands of patient samples each week. Existing analytical methods are challenging due to the complex nature of human plasma / serum matrices. In addition, to achieve the required detection limits, samples require complex liquid-liquid extraction techniques followed by derivatization. Traditionally, a protein precipitation step is used for fast cleanup of plasma samples. Protein precipitation is normally performed using a centrifuge tube or a 96-well collection plate. However, this process requires that supernatant be collected while being careful not to disrupt the pelleted protein in the bottom of the tube or collection plate. This step was greatly simplified by using Impact Protein Precipitation Plates. Three examples will be given in this poster. Vitamins and / or their metabolites were extracted from human plasma by performing a rapid protein precipitation using Impact Protein Precipitation Plates followed by HPLC analysis. HPLC analysis was performed using high efficiency HPLC columns using HPLC-UV, HPLC-FLD or LC/MS/MS systems. The Impact plate allows for the analysis of 96 samples at once, eliminates the transfer steps that are commonly associated with protein precipitation, and can also be automated. Protein precipitation was performed within the wells of the Impact plate and sample was not allowed to pass through the filter of the plate until vacuum was applied. This ensured that the precipitated protein was left within the wells of the Impact plate while protein-free sample was allowed to pass through the filter and into a collection plate (Figure 1). Impact technology offers easy, fast protein removal while providing maximized recovery of the target analytes. The HPLC columns used produced excellent chromatographic resolution, sensitivity, and high peak capacities for the analytes.

3 Figure 1. Protein precipitation using Impact Protein Precipitation Plates Proteins Target Analyte

4 Materials and Methods Vitamin C Protein Precipitation: Step 1. Place the Impact plate on top of a 96-well collection plate 2. Dispense 300 µl of cold 5 % Meta-phosphoric acid (4 C) into each well of the Impact plate 3. Add 100 µl of plasma/serum samples to each well of the Impact plate 4. Mix 5 times by aspirating with the same pipette tip 5. Centrifuge* the Impact plate at 500 g for 5 minutes at 4 C. Purified filtrate is collected in the 96-well collection plate. * A vacuum manifold may be used however 25 Hg vacuum pressure must be applied for up to 10 minutes or until sample is completely pulled through the Impact plate. After filtrate is collected, the collection plate containing the purified samples should be covered using a sealing mat. Filtrate was transferred to a Verex 9 mm screw-top amber autosampler vial to protect from light and was placed on a cooled autosampler (4 C). The sample is now ready to be immediately injected onto the LC/UV. HPLC Conditions: Column: Kinetex 5 µm XB-C18, 100 Å Gradient: Time (min) % B Dimensions: 150 x 4.6 mm 0 0 Part No.: 00F-4605-E Guard: SecurityGuard ULTRA UHPLC C Cartridges Cartridge Part No.: AJ Mobile Phase: A: 0.1 % Formic acid in water B: Acetonitrile Flow Rate: 800 µl/min Temperature: Ambient Detection: 245 nm Injection: 30 µl Vitamin B3 (Nicotinic acid and Nicotinamide) Protein Precipitation: Step 1. Place the Impact plate onto a suitable 96-well sample manifold 2. Dispense 300 µl acetonitrile into each well of the Impact plate 3. Add 100 µl of plasma/serum samples to each well of the Impact plate 4. Mix 3 times by aspirating with a pipette tip 5. Apply vacuum to filter the sample and collect the purified filtrate After filtrate is collected, the collection plate containing the purified samples should be covered using a sealing mat. The sample is now ready to be immediately injected onto the LC/MS/MS.

5 Materials and Methods (cont d) HPLC Conditions: Column: Gemini 3 µm C18 Dimensions: 100 x 4.6 mm Part No.: 00D-4439-E0 Mobile Phase: A: 0.1 % Formic acid in water B: Methanol Flow Rate: 600 µl/min MS/MS Conditions: An AB SCIEX API 4000 triple-quadrupole tandem mass spectrometer is used for analysis equipped with an ESI probe operating in positive polarity mode. Under MRM mode, two channels were monitored for nicotinamide and nicotinic acid. Peak Name Gradient: Time (min) % B Detection: API 4000 MS/MS, ESI Positive (ESI+) Temperature: Ambient Injection: 2 µl purified plasma MRM Channel Nicotinamide Nicotinic acid Vitamin B1 (TMP and Thiamine) Protein Precipitation: Step 1. Place the Impact plate onto a suitable 96-well sample manifold 2. Dispense 100 µl of plasma into each well of the Impact plate 3. Add 400 µl of methanol to each well of the Impact plate 4. Mix 3 times by aspirating with a pipette tip 5. Apply vacuum to filter the sample and collect the purified filtrate Transfer 50 µl to an autosampler vial (or collection plate). Add 50 µl of water and 50 µl of derivatizing reagent (15 % of sodium hydroxide solution plus 200 µl of 30 mm K 3 Fe(CN) 6 ). Cover the vial or collection plate with a lid or sealing mat, respectively. Vortex for 15 seconds. Put the vial or collection plate into an autosampler. Sample is ready to be injected onto the HPLC-FLD. HPLC Conditions: Column: Gemini NX-C18 3 µm Gradient: Time (min) % B Dimensions: 100 x 3.0 mm Part No.: 00D-4453-Y Mobile Phase: A: 25 mm Na 2 HPO 4, 10 % Methanol (ph 7.0) B: 25 mm Na 2 HPO 4, 70 % Methanol (ph 7.0) Flow Rate: 750 µl Temperature: 25 C Detection: Fluorescence (Excitation: 375 nm, Emission: 435 nm) Injection: 20 µl

6 Figure 2. Comparison of human plasma Vitamin C levels from extractions using 10 % Meta-phosphoric acid vs. 5 % Meta-phosphoric acid as a precipitation solvent % meta-pa 5 % meta-pa 80 Peak area Plasma-1 Plasma-2 Plasma-3 App ID 21447

7 Figure 3. Representative standard curve of Vitamin C at a concentration range of 0 to 100 µg/ml 3000 Vitamin C in Human Plasma by Impact Plate (Kinetex 5 µm, XB-C18, 150 x 4.6 mm + Guard) 2500 y = x R 2 = Peak area Vitamin C Concentration (µg/ml)

8 Table 1. Recovery of Vitamin C from spiked human plasma Added Vitamin C (µg/ml) Observed (µg/ml) Recovery (%) Mean 98.2 CV 4.8

9 Figure 4. Representative standard curve of nicotinic acid at a concentration range of 2 to 2000 ng/ml 1.5e6 1.4e6 R 2 = e6 1.2e6 1.1e6 1.0e6 9.0e5 Area, counts 8.0e5 7.0e5 6.0e5 5.0e5 4.0e5 3.0e5 2.0e5 1.0e5 0.0 App ID min Concentration, ng/ml

10 Table 2. Signal-to-noise ratio of nicotinamide and nicotinic acid at LOD and LOQ Analyte LOD LOQ ng/ml S/N Ratio ng/ml S/N Ratio Nicotinamide Nicotinic acid

11 Table 3. Recovery of TMP and Thiamine from human plasma after cleanup with Impact plates Recovery for Human Plasma - TMP Recovery for Human Plasma - Thiamine Added TMP Observed Recovery (%) Added Thiamine Observed Recovery (%) Mean 93.0 Mean 115.4

12 Table 4. Accuracy studies for TMP and Thiamine from human plasma after cleanup with Impact plates TMP-Spiked Plasma Controls Thiamine-Spiked Plasma Controls Control No. Expected Results Accuracy (%) Expected Results Accuracy (%) Mean Mean 88.9

13 Results and Discussion When developing a sample preparation procedure for vitamins and their metabolites in a high-throughput environment, it is important to choose an effective yet rapid method. Protein precipitation was chosen to prepare our plasma samples because it was rapid and could be automated using Impact Protein Precipitation Plates. Vitamin C Vitamin C is a very polar compound that is unstable at room temperature. Because of the instability of vitamin C we chose to screen various precipitating solvents in order to determine the most effective solvent for this work. It was determined that 5 % Meta-phosphoric acid yielded the highest recoveries of vitamin C as compared to other extraction solvents (Figure 2). Using the Impact plate to perform the precipitation step reduced the amount of time that our sample was exposed to room temperature as compared to processing by hand. After precipitation, vitamin C was analyzed by HPLC-UV. Vitamin C is a very polar compound and is difficult to retain via reversed phase HPLC; however, a Kinetex core-shell XB-C18 HPLC/UHPLC column provided a retention time of approximately 2.7 minutes, allowing us to accurately quantitate the target analyte. Using a wavelength of 245 nm, our analysis resulted in a correlation coefficient of and average recoveries of 98.2 (Figure 3 and Table 1). A lower limit of detection (LOD) of 0.78 µg/ml and lower limit of quantitation (LOQ) of 1.56 µg/ml showed a signal-to-noise (S/N) ratio of 14.6 and an average S/N ratio of Vitamin B3 (Nicotinic acid and Nicotinamide) Vitamin B3 exists as niacin (or nicotinic acid) as well as nicotinamide (a derivative of niacin), both of which are absorbed from the normal diet. Nicotinic acid is a light sensitive compound, making it important to ensure that it is prepared and handled with care. When analyzing plasma samples for nicotinic acid and nicotinamide, Impact plates were used because they allowed for high-throughput analysis, provided consistent recoveries, and reduced the amount of time spent performing the precipitation, which helped to keep the nicotinic acid stable. After protein precipitation, samples were immediately analyzed by LC/MS/MS using a Gemini 3 µm C18 HPLC column. The combined protein precipitation and LC/MS/ MS analysis resulted in a correlation coefficient of (Figure 4) and a signal-to-noise ratio of 4.3 and 3.3 at an LOD of 2 ng/ml and 12.4 and 10.1 at an LOQ of 10 ng/ml (Table 2). Vitamin B1 (TMP and Thiamine) Vitamin B1, also known as thiamine, is phosphorylated into thiamine monophosphate (TMP) in the mucosal cells of the duodenum. When analyzing human plasma samples for TMP and thiamine, Impact plates were used to perform a methanol precipitation. Using the plates provided rapid cleanup, saved time, and allowed this method to be easily automated for increased throughput. Following a protein precipitation step, the sample was derivatized and analyzed by HPLC-FLD using a Gemini NX- C18 3 µm HPLC column. Resulting recoveries of both target compounds averaged 93 % for TMP and 115 % for thiamine (Table 3). Accuracy studies resulted in an average accuracy of for TMP and 88.9 for thiamine (Table 4), suggesting that our method not only provided acceptable recoveries but was also accurate and reproducible.

14 Conclusion Vitamins are becoming a popular means to diagnose and treat disease states. With vitamin testing on the rise, laboratories must develop rapid, reliable, and robust analytical methods that can be easily incorporated into a high-throughput setting. Protein precipitation has always been a popular sample preparation method; however, the process can be improved upon. Using Impact Protein Precipitation Plates, several different vitamins and their metabolites were cleaned up from plasma using various precipitation solvents. The methods can also be automated, allowing laboratories to save time by increasing productivity while improving reproducibility and reducing the risk of analyte instability due to extended exposure to light or high temperatures. Preparation by automated protein precipitation is also a versatile sample preparation technique as the resulting extract can be analyzed by several different methods including HPLC/UV, LC/MS/MS, and HPLC-FLD. Trademarks Kinetex and Gemini are registered trademarks, Impact, SecurityGuard, and Verex are trademarks of Phenomenex. API 4000 is a trademark of AB SCIEX Pte. Ltd. AB SCIEX is being used under license. Gemini is patented by Phenomenex, U.S. Patent No. 7,563, Phenomenex, Inc. All rights reserved.