C146-E149 Technical Report vol.40 Internal Standard Addition During Pretreatment Using SIL-30AC The absolute calibration curve method is widely used for quantitation and requires that the standard solutions and sample solution be injected accurately at the same volume. When an internal standard method is used, quantitation is conducted based on the ratio of the internal standard substance (ISTD) to the analyte. Not only is correction made for the injection volume, but corrections can also be applied with respect to changes in the analysis environment, for example: the mobile phase flow rate, detector sensitivity, sample solvent evaporation, mobile phase composition, etc. Although there is more flexibility with injection volumes when using an internal standard method, high throughput analysis can be time consuming and error prone when analyzing many samples because the ISTD must be added to the sample beforehand. If the autosampler pretreatment functions are utilized, the ISTD can be automatically added to the sample right before it's injected. The internal standard method can be implemented in two ways, as a "stacked injection" or a "mixing and dilution injection." With the stacked injection, directly after the ISTD is aspirated into the needle, the sample is aspirated, and then both are injected into the column. This method is operationally convenient, as it allows for correction of fluctuating area values due to environmental changes including ambient temperature. With the internal standard mixing and dilution injection, not only is correction possible for environmental changes, injection volume errors can also be corrected because the ISTD and sample are first mixed in a separate vial. The selected ISTD must fulfill the following conditions. 1) Its peak must be completely separated from other constituent peaks in the sample. 2) Must not be present in the sample 3) Must elute in the vicinity of the analyte 4) Must have similar chemical structure as the analyte (not required if injection volume correction is intended) 2. Internal Standard Pretreatment Method Sample preparation, including "pretreatment" which includes dilution or mixing of multiple components for a pre-column reaction, is often performed by laboratory technicians before running batches of samples. It is the one major factor which can affect the overall time required and final results from the sample analysis, depending on skill of the technician preparing the samples. Since actual reaction times can differ slightly due to manual processing, and more time is usually required to conduct sample pretreatment manually, automating the pretreatment process can effectively improve analysis throughput and help to maintain repeatability of analysis results. Further, a small amount of manual intervention can result in large errors related to reaction operations, leading to much greater sample consumption as compared to autosampler-driven pretreatment. Another area in which automation is clearly indispensable is when conducting overnight primary screening of large numbers of samples. The SIL-30AC autosampler of the Nexera system is equipped with advanced pretreatment functionality as a standard feature, and includes rinsing options for the needle, loop, and injection port which reduce carryover to the greatest possible extent. Not only does this allow unattended analysis of large numbers of samples, highly reproducible data is obtained, especially for the analysis of derivatized samples, where results are easily affected by the accuracy of the reaction time following reagent addition to the samples. Here we introduce an automated internal standard addition pretreatment procedure, and examples of analysis repeatability. 1. Introduction C190-E140
Transcript
C146-E149
Technical Report vol .40
Internal Standard Addition During Pretreatment Using SIL-30AC
The absolute calibration curve method is widely used for quantitation and requires that the standard solutions and sample solution be injected accurately at the same volume. When an internal standard method is used, quantitation is conducted based on the ratio of the internal standard substance (ISTD) to the analyte. Not only is correction made for the injection volume, but corrections can also be applied with respect to changes in the analysis environment, for example: the mobile phase flow rate, detector sensitivity, sample solvent evaporation, mobile phase composition, etc. Although there is more flexibility with injection volumes when using an internal standard method, high throughput analysis can be time consuming and error prone when analyzing many samples because the ISTD must be added to the sample beforehand.If the autosampler pretreatment functions are utilized, the ISTD can be automatically added to the sample right before it's injected. The internal standard method can be implemented in two ways, as a "stacked injection" or a "mixing and dilution injection." With the stacked injection, directly after the ISTD is aspirated into the needle, the sample is aspirated, and then both are injected into the column. This method is operationally convenient, as it allows for correction of fluctuating area values due to environmental changes including ambient temperature. With the internal standard mixing and dilution injection, not only is correction possible for environmental changes, injection volume errors can also be corrected because the ISTD and sample are first mixed in a separate vial.The selected ISTD must fulfill the following conditions.1) Its peak must be completely separated from other constituent peaks in the sample. 2) Must not be present in the sample3) Must elute in the vicinity of the analyte4) Must have similar chemical structure as the analyte (not required if injection volume correction is intended)
2. Internal Standard Pretreatment Method
Sample preparation, including "pretreatment" which includes dilution or mixing of multiple components for a pre-column reaction, is often performed by laboratory technicians before running batches of samples. It is the one major factor which can affect the overall time required and final results from the sample analysis, depending on skill of the technician preparing the samples. Since actual reaction times can differ slightly due to manual processing, and more time is usually required to conduct sample pretreatment manually, automating the pretreatment process can effectively improve analysis throughput and help to maintain repeatability of analysis results. Further, a small amount of manual intervention can result in large errors related to reaction operations, leading to much greater sample consumption as compared to autosampler-driven pretreatment. Another area in which automation is clearly indispensable is when conducting overnight primary screening of large numbers of samples. The SIL-30AC autosampler of the Nexera system is equipped with advanced pretreatment functionality as a standard feature, and includes rinsing options for the needle, loop, and injection port which reduce carryover to the greatest possible extent. Not only does this allow unattended analysis of large numbers of samples, highly reproducible data is obtained, especially for the analysis of derivatized samples, where results are easily affected by the accuracy of the reaction time following reagent addition to the samples. Here we introduce an automated internal standard addition pretreatment procedure, and examples of analysis repeatability.
1. Introduction
C190-E140
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The pretreatment functions of the SIL-30AC are controlled through LabSolutions software. There are several preset templates available, and user-defined commands can also be programmed to allow more flexible operation. For example, commands can be entered that will perform extremely detailed operations such as "lower the needle 52 mm" into "vial number 1" and "aspirate 2 μL" at a rate of "15 μL/sec." The user-defined pretreatment programs are saved as part of the method for easy recall of the details of the pretreatment program from any data file acquired with that particular method.
3. Abundant Commands for Simplifying Pretreatment
Fig. 1: LabSolutions Pretreatment Function Setting Screen
4. Internal Standard Stacked Injection Pretreatment Method
The advantage of this method is that it allows for correction of the peak area value (intensity) to address changes in the environment (temperature, etc.). For example, when a sample is easily affected by temperature, an accurate concentration cannot be obtained when the peak area value changes due to temperature fluctuation, even if the injection volume is correct. In this situation, simultaneous analysis of the ISTD allows accurate calculation of the concentration from the ratio of analyte and ISTD area values because both the sample and the ISTD are subjected to the same fluctuations in environmental conditions. *However, the ISTD must have similar characteristics to the analyte.
4-1 Outl ine of ISTD Stacked Injection Operation Procedure
4-2 Internal Standard Stacked Injection Pretreatment Program (Example)
The respective volumes of ISTD and sample are aspirated sequentially, and injected directly onto the column. The operation procedure is as follows.
(1) ISTD is aspirated(2) Air is drawn (optional air gap)(3) Sample is aspirated(4) Injection and start of the time program
Assuming that the ISTD is in Vial No. 1 of Rack No. 0, injection of 1.0 μL using the ISTD stacked injection is programmed as shown in the following example.
Fig. 3: Internal Standard Stacked Injection Operation
Fig. 2: Temperature and Area RatioArea Ratio = 6/4 = 1.5 Area Ratio = 3/2 = 1.5
4
6
2
3
Temperature: 25 °C Temperature: 30 °C
The internal standard stacked injection allows accurate concentration calculation regardless of environmental changes.
ColumnISTD Analysis Sample
Needle
(1) (2) (3) (4)
: Moves needle to ISTD vial (Vial No. 1 of Rack No. 0): Lowers needle into ISTD vial: Aspirates 1.0 μL of ISTD: Raises needle and aspirates 0.1 μL of air: Immerses needle in rinse port to rinse needle: Moves needle to specified sample vial from the batch table or single run screen: Lowers needle into vial: Aspirates injection volume as specified in batch file: Returns needle to injection port, starts analysis: Ends pretreatment program
vial.n 0, 1n.strk nsaspir 1.0, ssair.a 0.1, ssd.rinsevial.n rn, sn
n.strk nsaspir iv, sss.injEnd
4-3 Example of Analysis Using Internal Standard Stacked Injection
An actual example of an analysis using the internal standard stacked injection method is as follows. For the ISTD, 40 μg/mL methylparaben was used, and the analyte consisted of ethylparaben at concentrations of 50 μg/mL, 100 μg/mL, 200 μg/mL and 400 μg/mL, respectively. Each sample was analyzed six times consecutively, and the area ratio repeatability with respect to the ISTD was determined.
The parameters [ss] and [rn], etc. in the program refer to the following.
: Needle stroke specified in method: Sample aspiration rate specified in method: Rack number specified for batch analysis or single run analysis: Vial number specified for batch analysis or single run analysis: Injection volume specified for batch analysis or single run analysis
nsssrnsniv
3
Fig. 4: Repeatability Using Internal Standard Stacked Injection(n=6, overlaid chromatograms)
Table 1: Repeatability Results with Internal Standard Stacked Injection
Concentration (μg/mL)Fig. 5: Calibration Curve (Internal Standard Stacked Injection)
Concentration (μg/mL)
50
100
200
400
0.248
0.212
0.148
0.201
0.0 0.5 1.0 1.5 2.0 2.5 min
Area Ratio (%RSD)
The advantage of this method is that any fluctuation in injection volume during the sample draw can be normalized. For example, even if only 9 μL is injected when the specified injection volume is 10 μL, the area ratio of the ISTD and analyte remains constant regardless of the actual volume injected, allowing correct calculation of the concentration.
5. Internal Standard Mixing and Dilution Injection Pretreatment Method
The first step is deciding which of the available solvents R0, R1, or R2 will be used as the sample diluent. The needle is then directed to the rinse port to "prime" or fill the sample loop and needle with the desired solvent before drawing the sample and ISTD. After aspirating the ISTD and sample at their specified volumes, they are dispensed into an empty vial along with the diluent that is already in the flow path from the solvent reservoir to the needle. After mixing in the designated vial, and refilling the needle and loop flow path with solvent R0 (initial conditions of the mobile phase in the method), the specified volume of the sample and ISTD mixture is aspirated and injected onto the column. The operation procedure is as follows.
(1) ISTD is aspirated(2) Air is drawn (optional air gap)(3) Sample is aspirated(4) ISTD, sample and diluent are dispensed together into an empty vial(5) Mixing is conducted(6) Sample is aspirated
5-1 Outl ine of Operational Procedure for the Internal Standard Mixing and Dilution Injection
Fig. 7: Internal Standard Mixing and Dilution Injection Operation
Fig. 6: Injection Volume Fluctuation and Area Ratio
Area Ratio = 10/1 = 10 Area Ratio = 9/0.9 = 10
1
10
0.9
9
Injection Volume: 10 μL Injection Volume: 9 μL
The internal standard mixing and dilution injection method offers correct concentration calculation even if the injection volume fluctuates.
: 40 °C: 254 nm: 1 μL each for ISTD and sample: 1: methylparaben 40 μg/mL (ISTD) 2: ethylparaben (50, 100, 200, 400 μg/mL)
Analytical Conditions
4
5-2 Internal Standard Mixing and Dilution Injection Pretreatment Program (Example)
The pretreatment program when using the internal standard mixing and dilution injection method is as follows.
n.drain : Moves needle to drain positiondisp 600,rs : Dispenses 600 μL of diluent from needle tip (to replace liquid in flow line)vial.n 0, 1 : Moves needle to ISTD vial at vial position No. 1 of Rack No. 0n.strk ns : Lowers needleaspir 10,ss : Aspirates 10 μLair.a 0.1,ss : Raises needle and aspirates 0.1 μL of air d.rinse : Moves needle to rinse port, immerses needle in rinse port to rinse needlea0 = sn+10 : Specifies sample vial position as 10 vial positions offset from mixing vialvial.n 1,a0 : Moves needle to Vial No. a0 sample vial of Rack No.1n.strk ns : Lowers needleaspir 10, ss : Aspirates 10 μLvial.n rn,sn : Moves needle to vial specified in the batch table or single run screen (empty vial for mixing)n.strk ns : Lowers needledisp 100,ss : dispenses 100 μL of diluent along with the 10 μL ISTD, 10 μL sample, 0.1 μL air mix 3,5,45,2,10 : Starts mixing process (3x mixing, 5 μL air aspirated, 45 μL sample aspirated, 2 μL/sec aspiration speed, 10 μL/sec dispensing speed)n.drain : Moves needle to drain portdisp 100,rs : Dispenses 100 μL diluent from needle tip to rinse interior of needled.rinse : Moves needle to rinse port, immerses needle in rinse port to rinse exterior of needleinj.p : Returns needle to injection portv.inj : Switches valve position to analysis flow line positionwait 2.0 : Keeps the loop and needle inline for 2 minutes for needle interior rinse by the pumpsgoto f0 : Goes to the file with the standard pretreatment program for sample injection End : Ends pretreatment operation
5-3 Analysis Example Using the Internal Standard Mixing and Di lution Pretreatment Method
*Designation of Vial Offset As shown in Fig. 8 below, if the vials containing sample (for example, No. 1−4) are spaced at the same distance from the empty mixing vials (for example, 11−14), samples can be pretreated and injected sequentially using a single pretreatment program with the variable a0 used to increment vials. This vial position scenario is specified in the program using the following expression:
a0 = sn + 10where: a0 is a variable to specify the sample vial number to be accessed during pretreatment sn is the empty vial number where mixing and the actual sample injection will take placeIn this example, the vials will be positioned as in the figure below.
An example describing an actual analysis using the dilution and mixing pretreatment method is presented below. For the ISTD, 400 μg/mL methylparaben was used, and for the analyte, ethylparaben was used at concentrations of 50 μg/mL, 100 μg/mL, 200 μg/mL, and 400 μg/mL, respectively. Instead of some volume of diluent present in the vial before the pretreatment program, one of the SIL-30AC rinse solutions was used for the diluent solution. Since the needle interior can be rinsed with up to 3 different solutions in the SIL-30AC, rinsing with two solutions is still possible even if one of the rinse lines is designated as the sample diluent. A dilution factor of 10 was used for both the ISTD and the samples. Each sample was analyzed six times consecutively, and the area ratio repeatability with respect to the ISTD was determined.The pretreatment program used for this analysis is shown below.
Fig. 8: Arrangement of Vials
Sample vials
No.11−14"Vial No." row in the
batch file is specified
using this No. 1−4
ISTD vial
No.1
Vial number: a0
Vial number: sn
Empty vials for mixing
No.1−4
Fig. 9: Injection Repeatability Using the Internal Standard Mixing and Dilution Pretreatment Method (n=6, overlaid chromatograms)
0.0 0.5 1.0 1.5 2.0 2.5 min
Eth
ylp
arab
en (1
0 μg
/mL)
ISTD
Column : Shim-pack XR-ODS (50 mmL. × 2 mmI.D., 2.2 μm)Mobile phase : A : Water / Acetonitrile = 75 / 25Flow rate : 0.5 mL/minColumn temperature : 40 °CDetection : 254 nmInjection volume : 5 μL of solution following dilution and mixing Peaks : 1: methylparaben 400 μg/mL (ISTD) 2: ethylparaben (50, 100, 200, 400 μg/mL)
Analytical Conditions
6. Example of Pretreatment Program for Amino Acid Analysis
A pretreatment program can be used to mix a reagent with the sample. Since the efficiency changes depending on the reaction time, and the pretreatment procedure determines the overall reaction time, the pretreatment operation can greatly contribute to the analysis repeatability. In the following example, a pretreatment method was used to perform the derivatization reaction of a food supplement amino acid (BCAA) with OPA ( -phthalaldehyde).
7. Conclusion
Data with excellent repeatability was obtained using both the internal standard "stacked injection" and the internal standard mixing and dilution pretreatment methods. The examples presented here focused on the use of the internal standard addition technique, but in the amino acid analysis example as well, the pretreatment program was utilized to demonstrate that good repeatability can be obtained without concern for even slight differences in reaction time. In particular, the more complicated the pretreatment process, the more difficult it becomes to maintain repeatability when conducting pretreatment manually. For this reason, the Nexera SIL-30AC pretreatment functions were designed to address a wide range of pretreatment processes, from automated primary screening of large numbers of samples to pretreatment requiring accurate reaction time.
*Concentrations indicate final concentrations following dilution.
Table 2: Repeatability Results with Internal Standard Mixing and Dilution Pretreatment Method
Concentration (μg/mL)
5
10
20
40
0.142
0.270
0.074
0.093
Area Ratio (%RSD)
Concentration (μg/mL)
Fig. 10: Calibration Curve (Internal Standard Mixing and Dilution Pretreatment Method)
Fig.11: OPA-Pre-Column Derivative Analysis Using SIL-30AC Pretreatment Function Template
R2 = 0.999988
0.000
0.200
0.400
0.600
0.800
1.000
1.200
0 5 10 15 20 25 30 35 40 45
0.0 1.0 2.0 3.0 4.0 5.0 min
Asp
SerGly
Thr
Arg
His
Ala
Tyr
Val
Met
Ile
Phe
Leu
Lys
Glu
Arg
Val
Ile Leu
Analysis of side-chain amino acid BCAA in food supplement
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