Rapid and sensitive measurement of cortisol and 6-Hydroxycortisol in human urine using LC-MS/MS.
Cytochrome P450 3A4 (CYP3A4) is the most abundant CYP metabolizing
enzyme. Cortisol can be converted to 6ß-hydroxycortisol (6ß-HC) by CYP 3A4. The latter is excreted in urine. Cortisol and 6ß-HC ratio in urine may act as CYP3A4 activity indicator.
- Human urine samples are spiked with internal standard and then extracted by liquid-liquid extraction with MTBE.
- Partial supernatant is dried, reconstituted, and injected into LC-MS/MS.
- STDs, LLOQ, and LQC are prepared in urine surrogate, PBS solution.
Column: Aquity BEH C18, 50 X 2.1 mm, 1.7 μm
Mobile Phase: 0.1% formic acid in water and ACN
Flow Rate: 0.5 mL/min
Mass Spec: Sciex API 5500, ESI+
|Processed stability||4 days||4 days|
|Ben-Top stability||18 hours||18 hours|
|Freeze/Thaw stability||4 cycles||4 cycles|
|Long-term stability||1 month||1 month|
A rapid and sensitive LC-MS/MS method was validated for cortisol and 6ß-HC analysis
in human urine. Method accuracy, precision, repeatability, selectivity, F/T stability, processed sample stability, bench-top stability, and long term stability have been confirmed.
Guodong (Gordon) Gu, Yifan Shi, and Yinghe Li
The plasma concentration ratio of 4b-hydroxycholesterol (4b-HC) to cholesterol has been recognized as a reliable marker for the assessment of Cytochrome P450 (CYP) 3A4 activity. It could be a valuable yet simple and cost effective side-product of a clinical study to evaluate CYP3A4-mediated drug-drug interactions.
Detecting 4b-HC in biological matrices using LC-MS/MS has been a challenge due to its poor ionization efficiency and lack of a predominant daughter ion, which causes a low sensitivity. Isomeric metabolites of cholesterol and other sterollike endogenous interferences also pose additional challenges for LC-MS/MS method development. In this study, a sensitive and robust LC-MS/MS method was developed and validated for the simultaneous determination of 4b-HC and cholesterol in human plasma.
The plasma sample (50 μL) was hydrolyzed using potassium hydroxide prior to the liquid-liquid extraction (LLE) with hexane. The extract of LLE was incubated for 30-minute with picolinic acid, then extracted again using hexane. The final extract was split to two portions and analyzed using LC-MS/MS with separate methods. 4b-HC-d7 and cholesterol-d7 were employed as the internal standards.
A nitrogen-containing moiety was introduced to 4b-HC and cholesterol molecules via a derivatization with picolinic acid. The enhanced ionization efficiency, and the formation of predominant product ions significantly increased the sensitivity of the SRM detection.
While the derivatization helped enhance the sensitivity, it significantly reduced the chromatographic baseline and eliminated the relevant interference peaks, which led to the successful chromatographic separation of 4b-HC within a 10-min run time. Five cholesterol metabolites, 4a-HC, 22-HC, 24-HC, 25-HC and 27-HC, were tested at 200 ng/mL for interference. None of these metabolites showed an interference peak at the retention time of 4b-HC, and the precision and accuracy of 4b-HC was not impacted.
Due to the high endogenous level of 4b-HC and cholesterol in blank plasma, a surrogate matrix (5% BSA) was used to prepare calibration standards. Precision and accuracy was evaluated by spiking known concentrations of analytes in the pre-quantified “blank” matrix.
The precision and accuracy of QC Samples were tested in three consecutive batches. The assay showed excellent linearity. The calibration curve was generated using a weighted 1/x2 regression. The endogenous concentrations of 4b-HC and cholesterol in more than 300 clinical samples were measured and revealed that 4b-HC ranged 12.5 -70.2 ng/mL, and cholesterol ranged 0.78 – 2.71 mg/mL.
- 4b-HC and cholesterol were quantified using only 50 μL of plasma samples in a semi-automated 96-
- The method was validated as linear, accurate,
precise and reproducible.
- The method has been successfully used to support several clinical studies
Yinghe Li, Brock Fiorito, Jean Liu, Meng Fang, Yifan Shi and Michael Zhang
The purpose of this study was to develop and validate an LC-MS/MS method for assaying N-acetylmannosamine (ManNAc) in human plasma (K2EDTA) to support a phase I clinical study.
GNE myopathy, previously known as hereditary inclusion body myopathy (HIBM) is an autosomal recessive, muscular disorder, characterized by progressive muscle weakness with onset in early adulthood. ManNAc is being investigated by NIH and New Zealand Pharmaceuticals, as the precursor of sialic acid, prevents the development of muscle disease in the mouse model of GNE myopathy.
As an endogenous compound, calibration standards for analyzing ManNAc in human plasma need to be prepared in surrogate matrix. The presence of multiple hydroxyl groups makes it difficult to use reversed-phase chromatography due to the lack of retention. Alliance Pharma developed a HILIC LC-MS/MS method with a calibration range of 10 to 5000 ng/mL. The method has been validated thoroughly to support clinical trials of ManNAc in GNE myopathy patients. The research was funded by NCATS via NCI Contract No. HHSN261200800001E.
ManNAc has four hydroxyl groups and relatively low molecular weight (221 amu). Its chromatography in various reverse phase columns had poor peak shape and low sensitivity. This problem was addressed by using an amide column in HILIC mode with trifluoroacetic acid (TFA) as the mobile phase modifier. The matrix background caused severe ion suppression which was probably due to the phospholipids in acetonitrile crashed human plasma. The suppression problem was solved by utilizing a phospholipid removal plate in the extraction step. Fifty microliters of human plasma was extracted with ManNAc-13C-d3 as the internal standard. No matrix effect was observed by comparing the results from QCs prepared in matrix and prepared in neat solution.
HPLC: Shimadzu LC-20AD
Column: Waters XBridge Amide, 100 x 2.1 mm, 3.5 µm
Mobile phase A: 0.2% acetic acid & 0.05% TFA in water
Mobile phase B: 0.2% acetic acid & 0.05% TFA in acetonitrile
Flow rate: 0.8 mL/min
Mass spectrometer: Sciex API 4000
Ionization mode: ESI positive
Source temperature: 400 °C
Ion transition monitored:
ManNAc: 222 → 126
ManNAc-13C-d3: 226 → 130
ManNAc Calibration Curve
The assay showed a linear calibration range of 10 to 5000 ng/mL. The curve was linear (R2 > 0.997) using weighted 1/x2.
Chromatogram and Sensitivity
Representative chromatograms of LLQC (10 ng/mL) and blank surrogate matrix (5% BSA). Retention time = 2.5 minute.
Precision and Accuracy of Spiked QCs
Spiked quality control sample precision and accuracy were demonstrated at n = 6 at the limit of detection (10 ng/mL) in one validation run, and at low (30 ng/mL), medium (251 ng/mL) and high concentrations (4051 ng/mL) over three validation runs.
ManNAc was spiked in the surrogate matrix as well as in plasma at n = 6. The recovery was calculated as the ratio of the mean peak area to that of the post-spiked sample. The recovery in 5% BSA was found to be 92.4%, while the recoveries in the plasma (medium and high QCs) were 132% and 113%. The fact that ManNAc is an endogenous compound probably led to recoveries over 100% for plasma samples, since the post-spiked level could only be estimated based on the endogenous concentration in plasma.
ManNAc Endogenous Concentration
Six lots of individual human plasma were quantified for the endogenous level of ManNAc. It ranged between 38.6 and 49.5 ng/mL. A large pool of plasma was assayed in six replicates (mean 50.5 ng/mL) and was used to prepared medium (200 ng/mL + endogenous level) and high (4000 ng/mL + endogenous level) QCs.
- A HILIC LC-MS/MS method capable of assaying ManNAc in human plasma was developed.
- As an endogenous compound, the calibration standards were prepared in surrogate matrix, while the QCs were prepared in surrogate matrix as well as human plasma.
- The method was demonstrated to be accurate, specific for ManNAc in human Plasma and reproducible.
- The assay has been successfully validated to support clinical trials of ManNAc in GNE myopathy patients.
Yifan Shi, Meng Fang, Michael Zhang, Yinghe Li, Amy Wang, Ed Kerns, Nuria Carrillo-Carrasco, Xin Xu, Selwyn Yorke, Bradley Gillespie
Alliance Pharma, Malvern, PA; TRND, NCATS, NIH, Rockville, MD; New Zealand Pharmaceuticals, Palmerston North, New Zealand
Leidos Biomedical Research Inc. (formerly SAIC-Frederick, Inc.), Frederick National Laboratory for Cancer Research, Frederick, MD
The purpose of this study was to develop and validate a rapid and sensitive LC-MS/MS method for measuring Goserelin in human plasma (K2EDTA).
Goserelin is a synthetic analogue of a naturally occurring luteinising-hormone releasing hormone (LHRH). With its ability to suppress production of sex hormones, Goserelin is particularly used in treatment of breast and prostate cancer. For identification and quantification of Goserelin in biological matrices, a method has been reported in rabbit plasma with a lower limit of quantification (LLOQ) of 0.1 ng/mL and an overall run time of 10 minutes. Due to the low level dosage of Goserelin, the challenge of analyzing and quantifying Goserelin at an even lower concentration has to be addressed. In this study, a rapid and sensitive HPLC-MS/MS method was developed and validated for the determination of Goserelin at an LLOQ of 40 pg/mL in human plasma.
In order to remove the complex interferences in matrix and enrich the analyte of interest, Waters Oasis WCX µElution plate was used to extract Goserelin and internal standard from human plasma. The human plasma samples were spiked with Triptorelin as internal standard and extracted using solid phase extraction. The eluent was evaporated to dryness and the residue was reconstituted with acetonitrile:water:formic aicd (10:90:0.5). The analysis was conducted utilizing the Schimadzu Prominence 20AC HPLC system coupled with SRM detection in ESI positive mode on a Sciex API5500 mass spectrometer. Chromatographic separation was achieved using a reverse phase column with 0.1% formic acid in water and 0.1% formic acid in acetonitrile as the mobile phases. The peak of interest was well separated from interference peaks within a 4.0 minute run time.
Short-term Stability and Reproducibility
Short-term stability of Goserelin in human plasma was established for 4 freeze/thaw cycles at -70oC/room temperature and 25 hours at room temperature. Reinjection reproducibility of the extracted samples was demonstrated by reinjecting standards and quality control samples stored at 6oC for 48 hours.
- A rapid and sensitive HPLC-MS/MS method for the quantification of Goserelin in human plasma was developed.
- Solid Phase Extraction was successfully used in order to remove the complex interferences in matrix and enrich the analyte of interest.
- The method was validated as linear, accurate, precise and reproducible. It can be used to determine the concentration of Goserelin in human plasma as low as 0.04 ng/mL using only 100 µL of sample.
Meng Fang, Yinghe Li, Yifan Shi
Alliance Pharma has successfully completed testing and validation of the Meso Scale Discovery® (MSD) Human V-PLEX Aβ Peptide Panel 1 Assay (6E10). With the completion of this validation, Alliance Pharma becomes the first global CRO to validate the Aβ V-PLEX Panel 1, and greatly expands its analytical capabilities in Alzheimer’s and Neurodegenerative Diseases.
The MSD Aβ V-PLEX Panel 1 (6E10) is a multiplexed sandwich immunoassay which measures three amyloid peptides of significance to Alzheimer’s disease: Aβ42, Aβ40, and Aβ38. MSD neurodegenerative disease assays provide a sensitive method for measuring the levels of peptide and protein targets within a single, small-volume sample. V-PLEX immunoassays are developed under rigorous design control and are fully validated according to “Fit for Purpose” principles.
Sensitive biomarker assays are proving crucial to the design and execution of clinical trials that target the earliest stages of Alzheimer’s disease. The Aβ Peptide Panel 1 (6E10) kit is validated for human CSF and thus is ideally suited for this purpose.
“The addition of the Meso Scale Discovery® MSD Aβ V-PLEX immunoassay to our service portfolio will further help our sponsors in the development of drugs to treat neurodegenerative diseases” said Dr. Feng Li, President of Alliance Pharma.
Years in business: 6
What made you start your business?
I got the idea a long time ago. I worked in same industry as I am now. I got intensive training. I appreciate the experience I got from that company. I was working very hard at learning and thinking about starting the business. By 2006, I began doing research on how to prepare for new business.
What did you do to survive the recession?
Alliance Pharmaceuticals was started in May 2008. We spent almost $1 million, however, only made about $28,000 with three key employees that year. We stuck with our initial business plan, focused on our expertise area, spent time to build up necessary functionalities and prepare for any potential business opportunities. All three employees deferred their pay for a year to ensure the business operation.
What has made your business successful?
We firmly adhere with our business philosophy including professional ethics, scientific excellence, regulatory compliance to win clients trust. We also have maximum business flexibility to understand our clients needs to meet and exceed their expectations.
What is the hardest part of your job?
The regulatory compliance to ensure the data quality and integrity and hiring the right employees.
What advice would you give someone starting their career or looking to start a business?
Have a dream, make a plan, prepare and execute it. You also must be confident enough to be persistent.
Would your advice be any different if you were addressing a minority?
There is no difference. The principle would be the same.
Excerpt from Philadelphia Business Journal
Circulating 25-hydroxyvitamin D3 (25-OHVD3) is widely accepted as the most useful biomarker for evaluating vitamin D status and diagnosing certain diseases.
Determination of 25-OHVD3 is important during some drug development since the increasing concern of these drugs potentially affecting vitamin D absorption.
Detecting 25-OHVD3 in biological matrix using LC-MS/MS has been challenged due to its poor ionization efficiency and lack of a predominant daughter ion, which causes a low sensitivity. Isomeric metabolites of vitamin D and other sterol-like endogenous interferences pose an additional challenge with chromatographic separation.
In this study, a sensitive and robust LC-MS/MS method was developed and validated for the determination of 25-OHVD3 in rat serum.
The samples were prepared in 96-well format by liquid-liquid extraction with MTBE, followed by a 30-minute derivatization with picolinic acid. 25-OHVD3-d6 was employed as internal standard.
Column: ACE C4, 100 X 4.6 mm, 3 µm particle size
Mobile Phase A: 0.1% formic acid in water
Mobile Phase B: 0.1% formic acid in acetonitrile
Gradient: 0-0.2 min, 75%B; 0.2-3.0min, 75-85%B; 3.05-5.50min, 95%B; 5.55-6.50min, 75%B
Flow rate: 1.5 mL/min
Detector: Sciex API 4000, ESI+
MRM transition: m/z 506.6 à 383.3 for 25-OHVD3
m/z 512.4 à 389.3 for 25-OHVD3-d6
A nitrogen-containing moiety was introduced to the 25-OHVD3 molecule via a derivatization reaction with picolinic acid to increase ionization efficiency (Figure 1).
Derivatization also ensured the formation of predominant product ions that can be used in SRM detections for both 25-OHVD3 and 25-OHVD3-d6 (internal standard) (Figure 2).
Due to high endogenous levels of 25-OHVD3 in blank serum, calibration standards were prepared in a surrogate matrix (5% BSA). Precision and accuracy was evaluated by spiking known concentrations of analyte in pre-quantified “blank” matrix.
Figure 3. Representative chromatograms of 25-OHVD3 and 25-OHVD3-d6 for LLOQ (0.5 ng/mL ) and in surrogate matrix and a rat serum sample showing endogenous 25-OHVD3.
This method was fully validated with a quantitation limit of 0.5 ng/mL and required only 50 mL of rat serum. The assay showed excellent linearity (R2>0.998) using a calibration range of 0.5 – 250 ng/mL (Figure 4).
The derivatization reaction with picolinic acid increased ionization efficiency of the 25-hydroxyvitamin D3 molecule and ensured the formation of predominant product ions, which in turn enhanced the LC-MS/MS sensitivity.
The method was validated as linear, accurate, precise and reproducible. It can be used to determine the concentration of 25-hydroxyvitamin D3 in rat serum as low as 0.5 ng/mL using only 50 mL of sample.
Yinghe Li, Yifan Shi, Meng Fang, and Pam Rogers
The purpose of this study was to develop and validate a LC-MS/MS method for assaying residual mercaptoethanol in in-process samples of a protein therapeutic product.
2-Mercaptoethanol is a widely used antioxidant in protein production and analysis. As a reducing agent, mercaptoethanol has the ability to cleave disulfide bonds, thus disrupting the tertiary and the quaternary structures of proteins. It is also included in enzyme solution to protect against catalytic site inactivation due to cysteine sulfhydryl oxidation or disulfide formation.
Mercaptoethanol is considered a toxicant, irritating the mucous membranes and respiratory tract, causing sore throat and abdominal pain, and potentially death if severe exposure occurs. As a result, determination of residual mercaptoethanol in biopharmaceutical products becomes crucial for patient safety.
Here, we report a LC-MS/MS method that utilizes derivatization with picolinic acid to quantitatively analyze residual mercaptoethanol and has been successfully used in biopharmaceutical manufacture.
The abundant presence of thiols in protein biopharmaceutical molecules posts the major challenge in analyzing residual mercaptoethanol. We chose LC-MS/MS technique to measure the specific molecule instead of traditional methods which usually only detect the thiol functional group. By derivatizing mercaptoethanol with picolinic acid to form the corresponding dipicolinyl ester (DPE), a detection limit of 4 parts per billion (ppb) was achieved.
The samples were extracted with ethyl acetate, derivatized with picolinic acid, and then extracted with hexane again for analysis. Mercaptoethanol-d4 was used as the internal standard.
HPLC: Shimadzu LC-20AD
Column: Agilent Zorbax SB-C18, 50 x 2.1 mm, 5 µm
Mobile phase A: 0.1% formic acid in water
Mobile phase B: Methanol
Flow rate: 0.6 mL/min
Mass spectrometer: Sciex API 4000
Ionization mode: ESI positive
Source temperature: 500 °C
Ion transition monitored:
Mercaptoethanol-DPE: 289 → 166
Mercaptoethanol-d4-DPE: 293 → 170
Precision and Accuracy of Spiked QCs
Spiked quality control sample precision and accuracy were demonstrated at n = 6 at the limit of detection (4 ppb) in one validation run, and at low (10 ppb), medium (200 ppb) and high concentrations (320 ppb) over three validation runs.
Mercaptoethanol was spiked in the matrix as well as in water at n = 6. The matrix effect was calculated as the difference of the mean peak area of the spiked matrix samples and the mean peak area of the pure samples. The method had virtually no matrix effect observed (2.4%).
The low molecular weight of mercaptoethanol (78 amu) affected selectivity and required careful selection of SRM transitions. The presence of thiol and hydroxyl groups makes it difficult to use reversed-phase chromatography due to the lack of retention. Low organic content during gradient HPLC elution leads to poor ionization efficiency and sensitivity. To address these challenges, mercaptoethanol was derivatized with picolinic acid to form a dipicolinyl ester, which a limit of detection at 4 ppb was successfully achieved.
- A novel LC-MS/MS method capable of assaying residue mercaptoethanol in in-process samples of a protein therapeutic product was developed and validated.
- Matrix interferences or unknown impurity peaks are readily removed by two steps of liquid-liquid extraction.
- Derivatization with picolinic acid increases the assay sensitivity to reach a detection limit of 4 ppb.
- The method was demonstrated to be accurate, specific for 2-mercaptoethanol and reproducible.
Yifan Shi, Yinghe Li, Meng Fang, William F. Wagner, Aston Liu, Sandro X. Nalli