Our paper aimed to develop rapid, sensitive, and specific LC-MS/MS method for the quantification of niacin (NA) and its metabolite nicotinuric acid (NUA) in human plasma. Following protein precipitation with acetonitrile, the NA, NUA, and internal standard (5-fluorouracil) were separated on a Zorbax 300SB-C8 column (250 mm × 4.6 mm, 5
Atherogenic dyslipidemia is highly prevalent, especially in patients with insulin resistance and diabetes mellitus. Atherogenic dyslipidemia increases the risk for coronary heart disease and peripheral vascular disease and remains a serious public health problem [
Various methods are available for determination of NA alone or NA along with its metabolites in biological fluids, like determination of NA and its metabolites by LC-MS/MS following deproteinization with acetonitrile in rat or human [
NA, NUA, and 5-FU were purchased from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). The purities of these compounds were found higher than 98%. Methanol and acetonitrile of HPLC grade were purchased from Fischer Scientific (Fair Lawn, NJ, USA). All other chemicals and solvents were of analytical grade. Milli-Q water used throughout the study was obtained from a Millipore system. Drug-free human plasma was provided by the General Hospital of Shenyang Military Area Command.
The mass spectrometer was API 4000 triple quadrupole system equipped with a TurboIonSpray ESI interface operated in negative multiple reaction monitoring (MRM) mode to monitor the transitions
Agilent 1100 system (Palo Alto, CA, USA) was used for solvent and sample delivery. The chromatographic separation for two analytes and IS was achieved by using a Zorbax 300SB-C8 column (250 mm × 4.6 mm, 5
Two separate stock solutions of the analytes were prepared at 1 mg/mL in methanol-water (50 : 50, v/v) and stored at −20°C. A series of standard working solutions for NA and NUA were prepared by diluting stock solution with methanol-water (50 : 50, v/v). The 5-FU solution was brought to a final concentration of 1000 ng/mL in acetonitrile from 1 mg/mL in acetonitrile. All working solutions were stored at 4°C and brought to room temperature before use. Calibration standards and quality control samples were prepared by spiking 50
Extraction of NA and NUA was carried out by protein precipitation. First, 50
Measurements for each analyte in the biological matrix should be validated according to relevant guidelines [
Selectivity is performed by using blank samples from at least six sources. Peak areas of endogenous compounds coeluting with the analytes should be less than 20% of the peak area of the LLOQ standard.
The lower limit of quantification (LLOQ) is considered being the lowest calibration standard and established using at least five samples. The calibration curves were defined in three separate runs on the basis of duplicate assays of the spiked plasma samples, and, on the same day, QC samples from three concentrations (Table
Precision and accuracy for the analysis of NA and NUA in quality control (QC) samples (
Concentration (ng/mL) | RSD (%) |
Relative | |||
---|---|---|---|---|---|
Added | Found | Intraday | Interday | ||
NA | 10.0 | 9.72 | 6.4 | 2.8 | −2.8 |
60.0 | 61.4 | 8.7 | 9.4 | 2.3 | |
600.0 | 587.3 | 5.0 | 7.4 | −2.2 | |
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NUA | 10.0 | 10.3 | 7.6 | 5.8 | 3.2 |
60.0 | 58.2 | 5.5 | 5.1 | −2.9 | |
600.0 | 596.0 | 5.7 | 3.7 | −0.6 |
Extraction recovery was measured at three levels by comparing the response of the analyte spiked before and after sample preparation. Matrix effects were assessed using a method similar to that reported by Matuszewski et al. (see below) [
Dilution integrity was evaluated by spiking the plasma with an analyte concentration above ULOQ and diluting this sample with blank plasma when processing. We investigated three concentrations by dilution of 20 times of QC samples.
Stability studies of NA and NUA should investigate the different storage conditions over time periods that equal or exceed those applied to the actual study samples. The following stability tests were evaluated: (1) stability of the stock solutions of the analytes; (2) freeze and thaw stability of the analytes in the plasma from −20°C to room temperature for three cycles; (3) short-term stability of the analytes in plasma at room temperature before extraction for 3 h and the ready-to-inject samples (after extraction, in the mobile phase) to the autosampler rack for 4 h; and (4) long-term stability of the analytes in matrix stored at −20°C for two months. All stability tests samples at three concentration levels (10, 60, and 600 ng/mL) were analyzed in triplicate and the deviations were determined in relation to freshly prepared samples.
The validated method was applied to the quantification of NA and its metabolite NUA in human plasma obtained from a clinical trial. It was in accordance with the Helsinki Declaration of 1975 (revised in 2008) [
Before conducting the study all subjects provided written informed consent to participate. The study was designed as randomized, open-label, crossover, and single dose periods with a 7-day washout between each treatment. A total of 12 healthy Chinese volunteers (6 males and 6 females) aged 20 to 35 years with a body mass index between 19 and 24 kg/m2 were recruited. Subjects were randomized into three groups (2 males and 2 females for each treatment). They were orally dosed with Niacin extended-release/Simvastatin 1, 2, and 3 tablets once daily, separately. The venous blood samples were collected at time intervals (0, 0.33, 0.66, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, 6.0, 8.0, 12, and 24 h) in separate vacutainers during the treatment. All blood samples were centrifuged at 3000 ×g for 10 min and the plasma was separated and kept frozen at −20°C until analysis.
The pharmacokinetic parameters were calculated with data analysis system (DAS) program (version 2.0, Mathematical Pharmacology Professional Committee, Shanghai, China). The maximum plasma concentration (
Statistical analysis of pharmacokinetic parameters was performed using SAS version 9.1 (SAS Institute Inc., Cary, North Carolina, USA). A
We used ESI-MS/MS to analyze the compounds. To quantify the analytes using the MRM mode, the full scan and product ion spectra of the analytes and IS were investigated. Owing to the similar functional group pyridine ring and carboxyl group of NA and NUA, it makes them give excellent sensitivity in the negative ESI mode. The deprotonated peak was the predominant form of the molecular ion for NA at
Product ion mass spectra of the deprotonated molecules of NA (a), NUA (b), and 5-FU (c).
In order to optimize the LC system for the detection of NA and NUA, chromatographic separation was tested on several C18 and C8 columns to achieve the best efficiency and peak shape: Phenomenex Gemini (150 mm × 2.0 mm, 5
We have chosen 5-FU as an internal standard. Although a stable isotope-labeled compound would be an ideal IS for quantitation in complex matrices in LC-MS/MS analysis, it is not readily available in most laboratories. Hence, structural analogues were screened to find suitable compound for use as IS. 5-FU was finally selected as the IS for the determination of NA and NUA.
Prior to loading the sample for LC injection, the coextracted proteins should be removed from the prepared solution. Different extraction procedures like protein precipitation (PPT), liquid-liquid extraction (LLE), and solid phase extraction (SPE) were tested. The largely polar character of NA and NUA makes it difficult to extract from plasma with organic solvents. In this study, NA and NUA were prepared by plasma protein precipitation. Moreover, when acetonitrile was chosen for protein precipitation, the extraction recovery was higher when methanol was used. A reconstitution procedure for the dried extract employed that optimized recovery of all analytes while at the same time sufficiently reduced the presence of endogenous matrices.
Method specificity was demonstrated by comparing the MRM chromatograms of blank samples with those of spiked samples. No interference was detected from endogenous substances within the analytes and IS (Figure
Representative MRM chromatograms for NA (I), NUA (II), and IS 5-FU (III) in human plasma: (a) blank plasma sample; (b) blank plasma sample spiked with NA and NUA at the lowest limit of quantification (5 ng/mL) and IS (1000 ng/mL); (c) plasma sample obtained from a subject 12 h after oral administration of 3 tablets of Niacin extended-release/Simvastatin.
The linear ranges were both 5–800 ng/mL for NA and for NUA in the human plasma with correlation coefficients of >0.995. The lower limits of quantification, defined as the lowest concentration measured with ±15% accuracy and ≤15% precision, were both 5 ng/mL for NA with relative errors 6.1% and NUA with relative errors 0.7%.
Assay precision and accuracy were determined by using QC samples at three concentrations in replicates (
The average extraction recoveries for NA were
Coeluting matrix compounds, in the plasma samples, may reduce or enhance the ion intensity of the analytes and affect the reproducibility and accuracy of the assay. The matrix effect was assessed as follows: to the blank matrix from six different individuals, analytes were added at three concentrations (low, middle, and high), making a total of 18 samples. These samples were subjected to the analytical procedure and compared with the standard working solutions.
The relative matrix effect of NA was 80.0–84.4, 83.1–96.5, and 80.8–91.2% at the concentrations of 10, 60, and 600 ng/mL, respectively. The relative matrix effect of NUA was 81.7–96.2, 85.3–99.3, and 83.5–96.6% at the concentrations of 10, 60, and 600 ng/mL, respectively. It indicated that the matrix effects had no practical effect on the quantification of NA and NUA.
The relative errors for QC samples of 20-fold dilution with control matrix for NA were from −3.7% to 1.2% and 11.7% to 2.4% for NUA. The results demonstrated that plasma samples could be diluted 20-fold with control matrix with no effect on the accurate quantitation of NA and NUA.
The stability of NA and NUA in human plasma was investigated under a variety of storage and process conditions. The results of the stability studies (Tables
Stability data for stock solutions of NA and NUA 6 h in room temperature and 7 days at −20°C (before and after).
Response (×105) | |||
---|---|---|---|
Before | Six h at room temperature | Seven days at −20°C | |
NA | 14.7 | 14.6 | 13.9 |
15.5 | 15.6 | 15.4 | |
14.2 | 14.1 | 13.9 | |
14.0 | 15.8 | 14.2 | |
14.7 | 15.1 | 14.3 | |
16.9 | 13.8 | 13.1 | |
R.E.% | −1.1 | −5.8 | |
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NUA | 6.0 | 6.8 | 6.1 |
6.6 | 6.5 | 6.3 | |
6.3 | 6.5 | 6.4 | |
6.4 | 6.7 | 6.4 | |
6.5 | 6.3 | 6.6 | |
7.0 | 6.3 | 6.5 | |
R.E.% | 0.8 | −1.6 |
Stability data for NA and NUA in human plasma under different storage conditions (before and after analysis,
Storage conditions | Added |
Before |
Found |
|
---|---|---|---|---|
(ng/mL) | (ng/mL)/% | (ng/mL)/% | ||
NA | Short-term |
10.0 | 9.4/−5.8 | 9.1/−8.9 |
60.0 | 60.2/0.4 | 58.8/−2.1 | ||
600.0 | 611.0/2.17 | 636.3/6.0 | ||
Long-term |
10.0 | 9.4/−5.7 | 9.6/−4.1 | |
60.0 | 56.0/−6.6 | 53.7/−10.6 | ||
600.0 | 527.0/−12.2 | 595.3/−0.8 | ||
Three freeze/thaw cycles | 10.0 | 9.4/−1.9 | 9.5/−4.6 | |
60.0 | 60.2/0.4 | 58.0/−3.28 | ||
600.0 | 611.0/1.8 | 546.3/−9.0 | ||
Autosampler for 4 h (24°C) | 10.0 | 9.9/−1.0 | 9.6/−3.7 | |
60.0 | 61.6/2.6 | 62.7/4.5 | ||
600.0 | 597.4/0.4 | 578.3/−3.6 | ||
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NUA | Short-term |
10.0 | 10.8/7.7 | 11.0/9.7 |
60.0 | 60.1/0.2 | 63.6/5.9 | ||
600.0 | 624.7/4.1 | 628.7/4.8 | ||
Long-term |
10.0 | 10.2/2.3 | 10.6/6.4 | |
60.0 | 60.5/0.8 | 59.1/−1.5 | ||
600.0 | 608.0/1.3 | 579.3/−3.4 | ||
Three freeze/thaw cycles | 10.0 | 10.8/7.7 | 10.8/8.3 | |
60.0 | 60.1/0.2 | 63.3/5.6 | ||
600.0 | 624.7/4.1 | 618.3/3.1 | ||
Autosampler for 4 h (24°C) | 10.0 | 10.9/9.3 | 10.7/7.3 | |
60.0 | 59.4/−0.9 | 67.0/11.7 | ||
600.0 | 611.3/1.9 | 598.3/−0.3 |
After validation, the LC-MS/MS method was used to determine NA and NUA concentrations in plasma samples after oral administration of Niacin extended-release/Simvastatin to two groups’ healthy volunteers. The results were summarized in Table
The main pharmacokinetic parameters for NA and NUA after single oral doses of Niacin extended-release/Simvastatin to healthy volunteers (mean ± S.D.,
1 tablet | 2 tablets | 3 tablets | ||||
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NA | NUA | NA | NUA | NA | NUA | |
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0.3846 ± 0.1917 | 2.151 ± 1.362 | 1.538 ± 0.9570 | 5.396 ± 3.215 | 6.021 ± 6.579 | 10.21 ± 7.076 |
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0.5176 ± 0.2265 | 2.181 ± 1.368 | 1.719 ± 0.9230 | 5.478 ± 3.249 | 6.097 ± 6.555 | 10.25 ± 7.091 |
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8.80 ± 8.49 | 1.70 ± 0.801 | 7.33 ± 8.68 | 2.55 ± 1.26 | 5.40 ± 4.57 | 2.72 ± 1.83 |
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0.19 ± 0.20 | 0.53 ± 0.31 | 0.43 ± 0.69 | 0.33 ± 0.14 | 0.27 ± 0.25 | 0.33 ± 0.15 |
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5.37 ± 1.96 | 2.91 ± 0.74 | 4.40 ± 2.21 | 3.60 ± 1.32 | 3.65 ± 1.33 | 3.79 ± 1.11 |
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0.1598 ± 0.1253 | 0.8360 ± 0.3989 | 0.9040 ± 0.4870 | 1.628 ± 0.6360 | 2.476 ± 1.431 | 2.794 ± 1.038 |
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2.94 ± 2.63 | 2.06 ± 1.60 | 1.82 ± 1.69 | 3.03 ± 2.19 | 2.42 ± 1.64 | 2.33 ± 1.05 |
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1271 ± 850.0 | 287.8 ± 128.2 | 744.0 ± 356.0 | 224.8 ± 91.90 | 526.0 ± 421.0 | 189.3 ± 94.20 |
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13.71 ± 11.60 | 0.6490 ± 0.3380 | 7.649 ± 9.324 | 0.8210 ± 0.5230 | 5.199 ± 6.651 | 0.6960 ± 0.4580 |
Figure
Mean plasma concentration-time profiles and logarithmic transformation profiles of NA (a) and NUA (b) after oral single dose administration of Niacin extended-release/Simvastatin (1 tablet 500 mg/10 mg, 2 tablets 1000 mg/20 mg, and 3 tablets 1500 mg/30 mg) to volunteers (
We found that there was significant difference between subjects, while there was no significant statistical difference (
In this study, the LC-MS/MS method were developed and validated for the quantification of NA and its active metabolite NUA in human plasma. The method has been successfully applied to pharmacokinetic study after oral administration of a fixed dose combination tablet of Niacin extended-release/Simvastatin (500 mg/10 mg) to the humans. The results showed that the plasma concentration and pharmacokinetic performance of NA and NUA have significant difference between individuals. It indicated that much attention should be paid during the clinical use especially in coadministration when suffering from several illnesses. Our results have clinical implications and warrant further investigation of Niacin extended-release/Simvastatin.
The authors declare that they have no conflict of interests.
This study was supported by the Key Project of the National 12th Five-Year Research Program of China (no. 2012ZX09303016-002).