How to Calculate Spiked Recovery Rate in Chromatographic Analysis
Introduction
In analytical chemistry, spiked recovery rate is a fundamental parameter used to evaluate the accuracy of an analytical method. It reflects how effectively the target analyte can be recovered from a sample matrix during the entire sample preparation and analysis process. In HPLC, GC, LC-MS, and other instrumental analysis techniques, spiked recovery experiments are widely applied during method validation to verify whether sample extraction, purification, and detection procedures introduce significant analyte loss or matrix interference.
What is Spiked Recovery Rate?
The spiked recovery rate refers to the ratio between the measured amount of a target compound recovered from a sample after adding a known amount of standard substance versus the theoretical amount that was added.
In a typical recovery experiment, a known quantity of analyte standard is added into a sample matrix, and the spiked sample is processed using the same pretreatment and analytical procedures as the actual sample. The measured result is then compared with the theoretical added amount to calculate the recovery rate. The calculation formula of spiked recovery rate is: P (%) = [(Spiked Sample Value – Sample Value) / Spike Value] × 100%.
A recovery rate close to 100% indicates that the analytical method can accurately extract and determine the target analyte from the sample matrix.
The Three Faces of Spiked Recovery
The context of an experiment dictates the type of test to be employed. Generally, spiked recovery experiments can be divided into three categories.
Blank Spiked Recovery: In this type of test, a blank matrix (a sample that does not contain the target analyte at all) is used. A known amount of standard substance is added to this blank sample, and processed through all analytical steps. The ratio of measured analyte to theoretical spiked mass defines the blank recovery rate. It is mainly used to evaluate the accuracy of the analytical procedure itself.
Negative Sample Spiked Recovery: A negative sample is a sample matrix that does not contain detectable levels of the target analyte. In this type of test, two identical portions of the negative sample are prepared: one of them is analyzed directly without adding standard substance, and the other is spiked with a known amount of target standard substance. Both are analyzed under identical conditions. The recovery is calculated by subtracting the control's response from the spiked sample's response, then divided by the theoretical added amount.
Positive Sample Spiked Recovery: A positive sample already contains a certain amount of the target analyte. The procedure is similar to negative sample spiking, but the original analyte concentration in the sample must be determined by a preliminary analysis. The spike amount is then chosen (which is typically equivalent to the original concentration).
Practical Guidelines for A Valid Spiked Recovery Experiment
The most common pitfalls in recovery experiments are not in the math, but in the physical execution. Here are some common guides for obtaining accurate, reliable recovery data.
- Match the physical and chemical form of the added standard and that of the target analyte.
- Match the spike amount with the sample content. Typically, the spike amount in mass is between 0.5× to 2× the original analyte mass, and not exceed 3×. The concentration after spiking must not exceed the upper limit of the method. If the original sample concentration is below the limit of detection (LOD), spike at the LOD.
- Make the spike concentration high, and volume as small as possible. The volume of the spiking solution should be no more than 1% of the original sample volume. If the analyte concentration in the sample is close to the method LOD, the spiking level should be limited to the low concentration range of the calibration curve.
- After spiking, the measured concentration should not exceed 90% of the upper limit of the analytical method.
Calculating the Required Spiking Amount
Suppose we are working with a method whose sample pretreatment is as follows: Weigh 5 ± 0.01 grams of the sample, place in a 50 mL centrifuge tube, add 10 mL of acetonitrile, vortex for 2 minutes, sonicate for 15 minutes, and centrifuge at 4000 r/min, 4 °C for 5 minutes. Transfer the supernatant to a 25 mL colorimetric tube, and extract the residue with another 10 mL of acetonitrile. Combine the extract into the colorimetric tube and dilute with acetonitrile to the mark. Mix well. Measure 5 mL of the extract through the SPE cartridge, collect the eluate, nitrogen evaporate at a 40 °C water bath to dryness, dilute to 1 mL with methanol, and filter through a 0.22 µm membrane.
Assume the final required instrumental concentration is 10 ng/mL, and the stock standard solution of the target analyte has a concentration of 1 mg/L. How do we calculate the required spike amount?
- Step 1: Determine the final amount. The final solution volume before injection is 1 mL, therefore, the amount of analyte required in the final solution is n = C × V = 10 ng/mL × 1 mL = 10 ng.
- Step 2: Calculate the amount before dilution during sample preparation. Only 5 mL out of the 25 mL extract volume is taken for SPE cleanup. That is, only 5 mL/25 mL = 1/5 of the extracted analyte enters the final solution. Therefore, the initial added standard is 5 times the final amount: 10 ng × 5 = 50 ng.
- Step 3: Calculate the volume of standard solution required. The concentration of the stock standard solution is 1 mg/L, or 1000 ng/mL. Therefore, the required volume is V = n / C = 50 ng / 1000 ng/mL = 0.05 mL = 50 µL.
In practical calculations, the exact sample weighing value should be used instead of the nominal 5 g. In addition, the purity of the standard substance should also be considered and corrected during calculation.