Archive March 2019

A Patient’s Guide to Psoriatic Arthritis

A Patient’s Guide to Psoriatic Arthritis

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Arthritis is an umbrella term that simply describes joint inflammation. There are more than 100 types of arthritis, and they all can have different triggers and characteristics. As such, arthritis is one of the most common chronic conditions, affecting about 54 million Americans or nearly a quarter of all adults. But not all forms of arthritis are created equal. Some, like psoriatic arthritis, are related to immune disorders and can cause a lot of pain and disability. What Is Psoriatic Arthritis?
Psoriatic arthritis is one of several inflammatory forms of arthritis, and it’s related to the common skin disorder psoriasis. Psoriasis is a chronic skin problem that speeds up the life cycle of skin cells. This causes a rapid build-up of cells on the surface of the skin that collect in red, scaly patches. It can be very itchy and painful.
The Arthritis Foundation reports that about 30 percent of people with psoriasis “also develop a form of inflammatory arthritis called psoriatic arthritis.” Like psoriasis, psoriatic arthritis is an autoimmune disorder, meaning that the immune system begins attacking the body’s normal cells, causing inflammation, swelling, pain and eventually damage to the joints.
Dr. David Pugliese, a rheumatologist at Geisinger in Danville, Pennsylvania, says that psoriatic arthritis is one of “a whole host of autoimmune conditions,” that cause joint pain, swelling and other complications. “These conditions can give people arthritis and come with their own host of immune concerns that we need to monitor,” Pugliese says, so it’s important to speak with your doctor if you notice any arthritis-like symptoms in any of your joints. Types of Psoriatic Arthritis
Within psoriatic arthritis, there are three main types. Symmetric psoriatic arthritis affects about 50 percent of patients, and it occurs in joints on both sides of the body. Asymmetric psoriatic arthritis affects about 35 percent of people with psoriatiac arthritis. It’s generally considered the mildest form and tends to affect three or fewer joints. Three other types of psoriatic arthritis make up the rest of cases. Arthritis mutilans occurs in the fingers and toes and is an advanced, deforming type of psoriatic arthritis. Spondylitis impacts the shoulders and neck. And distant interphalangeal predominant psoriatic arthritis impacts the joints at the ends of the fingers. Symptoms
The Arthritis Foundation reports that “most people with psoriatic arthritis have skin symptoms before joint symptoms.” Skin symptoms of psoriasis include: Red patches of raised or inflamed skin. Whitish or silvery scales or plaques on those red patches. Dry skin that cracks or bleeds. Tenderness, itching or burning around the site of the patches. Thick, pitted or ridged nails. Swollen and stiff joints.
But for some people, psoriatic arthritis can develop in the absence of any skin symptoms. “You don’t have to have psoriasis to have psoriatic arthritis,” says Dr. Esther Lipstein-Kresch, chief of rheumatology at ProHEALTH Care in New York. That can sometimes make diagnosis difficult, as the symptoms that accompany psoriatic arthritis may sometimes be similar to those of other forms of inflammatory arthritis.
Common symptoms of psoriatic arthritis include: Swollen, painful joints. Joint stiffness, especially first thing in the morning. Sausage-like swelling along the length of the fingers or toes. Tendon or ligament pain. Skin rashes and pitted nails. Fatigue. Redness and irritation of the eyes and distorted vision.
One of the key distinguishing features of psoriatic arthritis is the development of “sausage-shaped deformity,” Lipstein-Kresch says. “It can happen in the toes and fingers,” and it can be very uncomfortable. This particular type of swelling is less likely to occur in rheumatoid arthritis so it’s often a differentiator in helping your doctor make the right diagnosis.
Another feature of psoriatic arthritis is the development of bone fusions, “where the bones literally fuse together,” Lipstein-Kresch says, a condition called ankylosis. This usually only happens in severe cases that aren’t well managed. “If you’re unlucky enough to fuse your wrist in a really bad position when it’s flexed, you wind up like that the rest of your life. Sometimes people fuse in a neutral position, which is good because if it’s fused, it doesn’t hurt that much and they can still use their hands .” In fact, surgical fusion is sometimes used as a treatment for psoriatic arthritis and other inflammatory arthritis conditions.
While ankyloses can also occur in other inflammatory arthritis conditions, such as rheumatoid arthritis, “psoriatic arthritis generally has more fusion than rheumatoid arthritis. But hopefully in this day and age with all these new biologic medications and other disease-modifying drugs, people wouldn’t get fusion,” Lipstein-Kresch says. Diagnosis
To diagnose a case of psoriatic arthritis, your doctor will conduct a thorough physical exam and take an extensive medical history. Your rheumatologist may also order some blood tests to gauge how much inflammation you have in your body or X-ray images of your symptomatic joints to take a look at what’s going on inside them. You may also have MRIs, ultrasounds or CT scans to look at the extent of damage in your joints.
Although there isn’t a specific blood test currently in use that can say definitively that you have psoriatic arthritis, your rheumatologist will likely order some blood tests to rule out other forms of inflammatory arthritis, such as rheumatoid arthritis. These tests look for an antibody in the blood called the rheumatoid factor. Even if your RF test comes back negative, it doesn’t necessarily mean you don’t have rheumatoid arthritis. Lipstein-Kresch says that about 85 percent of people with rheumatoid arthritis will have a positive RF factor test but “the other 15 percent won’t. Those people have what’s called zero-negative RA.”
Even a positive RF test doesn’t completely rule out a diagnosis of psoriatic arthritis, as the two conditions can coexist, although the National Psoriasis Foundation reports this coexistence of conditions is rare. Nevertheless, it’s important to work with a doctor who has experience in treating arthritis conditions to be sure you’re arriving at the right diagnosis. “Even though we have some tools we use in terms of immunological testing that are helpful, ultimately (the correct diagnosis) still boils down to what the patient looks like on exam and what kind of story they are telling,” Pugliese says. Causes and Risk Factors
Psoriatic arthritis occurs when the body’s immune system misidentifies normal cells as foreign invaders and goes on the attack. The National Psoriasis Foundation reports that skin symptoms are present before the development of joint pain in about 85 percent of cases.
It’s not entirely clear why psoriatic arthritis develops in some people and not others, but genetics are likely part of the answer, as well as environmental factors. People with a family history of either psoriasis or psoriatic arthritis are at higher risk of developing the disorder.
It’s believed that physical trauma or certain environmental factors, such as the introduction of a bacterial or viral infection, could sometimes trigger the immune response that develops into psoriatic arthritis in some people.
Age may also be a risk factor. Although anyone can develop psoriatic arthritis at any age, the Mayo Clinic reports that “it occurs most often in adults between the ages of 30 and 50.” Treatment
Although there’s currently no cure for psoriatic arthritis, the condition can be managed. Patients may be offered a range of different medications, including: Nonsteroidal anti-inflammatory drugs (NSAIDs). NSAIDs reduce inflammation and relieve pain. Some, such as ibuprofen (Advil, Motrin IB, etc.) and naproxen sodium (Aleve) are available over the counter . Your doctor may prescribe stronger NSAIDs to improve symptoms of psoriatic arthritis. Disease-modifying antirheumatic drugs (DMARDs). These drugs stall the progression of psoriatic arthritis and may be used to treat other forms of inflammatory arthritis. Methotrexate (Trexall), leflunomide (Arava) and sulfasalazine (Azulfidine) are three common DMARDs used to treat psoriatic arthritis. Steroids. Powerful anti-inflammatories, steroids such as prednisone (Deltasone, Rayos and Prednisone Intensol) can help bring down a flare-up of psoriatic arthritis or other inflammatory arthritis disorders quickly. Steroids have been around a long time, and “are still excellent medications in terms of cooling things down,” Lipstein-Kresch says. “When you see somebody who is really inflamed and has a lot of pain, can’t sleep and can’t function, the steroids make a tremendous difference very rapidly.” But she says these are generally used sparingly as they can have some harsh side effects when used long-term. Steroids may also be used in combination with other medications to help prime the body’s response for the effects of biologic or other medications. Immunosuppressants. In immune disorders, the body’s natural defense mechanism – the immune system – works overtime and on the wrong targets. By tamping down some of this response, some patients may find some relief from autoimmune conditions such as psoriatic arthritis. Azathioprine (Imuran, Azasan) and cyclosporine (Gengraf, Neoral, Sandimmune) are two immunosuppressant drugs that may be used to treat the condition. Biologics. In the past decade, new medications have emerged to help patients deal with a number of inflammatory arthritis conditions. The NPF reports that these types of drugs are administered by injection or intravenous infusion and are derived from living cells cultured in a lab. Among these are: TNF-alpha inhibitors. These medications target tumor necrosis factor-alpha, an inflammatory substance produced by the body. By inhibiting the production of this substance, that may lead to reduced pain and stiffness and relief from joint swelling. Etanercept (Enbrel), infliximab (Remicade) and adalimumab (Humira), are three examples of this class of drug. Interleukin inhibitors. These drugs selectively target certain cytokines (a kind of protein) involved in inflammatory and immune responses. Examples include secukinumab (Cosentyx), ixekizumab (Taltz), brodalumab (Siliq) and ustekinumab (Stelara). T-Cell inhibitors. T-cells are a kind of white blood cell that create inflammation in psoriatic disease. The drug abatacept (Orencia) may help tamp down some of this response.
Few patients progress to the point of needing surgery, but it can be an option in some cases. In a procedure called synovectomy, your doctor might remove the damaged or diseased tissue from inside the joint. In other cases, a patient may undergo a joint replacement surgery, joint resurfacing or realignment surgery or a bone fusion to relieve pain in a problematic joint. Management
Another hallmark of psoriatic arthritis is frequent flare-ups, or periods when the disease seems worse, followed by periods of fewer symptoms. Managing this ebb and flow is an important part of living with the disease.
Because autoimmune disorders are so closely associated with excessive levels of inflammation in the body, some doctors and patients believe that diet can impact the severity of symptoms . “There are a million and one diets you could find online, and some people come in and tell me every time they have something fried, their arthritis flares up. Or they gave up gluten and feel better, even though there’s no indication that they actually have a gluten sensitivity,” Lipstein-Kresch says. It’s hard to know exactly what might help most in which situation, so it’s best to talk with your doctor about what’s best for you.
In general, it’s believed that foods high in omega-3 fatty acids, such as fish, healthy oils and nuts, could help reduce inflammation in the body. A diet rich in brightly colored fruits and vegetables will also provide lots of antioxidants, which may also help control inflammation. Some studies have connected red meat, high-fat and high-cholesterol foods with increased inflammation, but Lipstein-Kresch says the evidence is “soft” and her best advice is to “eat healthy.”
Exercising regularly is also typically recommended to keep your joints moving as smoothly as possible, retain as much range of motion as possible and to keep your weight in check. When your body is strong, you’ll be better able to cope with your condition. Best Rheumatology Hospitals

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A Simple Ultraperformance Liquid Chromatography-Tandem Mass Spectrometry Method for Measurement of Cortisol Level in Human Saliva

<h1>A Simple Ultraperformance Liquid Chromatography-Tandem Mass Spectrometry Method for Measurement of Cortisol Level in Human Saliva</h1>

A Simple Ultraperformance Liquid Chromatography-Tandem Mass Spectrometry Method for Measurement of Cortisol Level in Human Saliva

Correspondence should be addressed to Muhammad M. Hammami ;
Received 10 October 2018; Revised 20 December 2018; Accepted 15 January 2019; Published 3 March 2019
Academic Editor: Barbara Bojko
Copyright © 2019 Syed N. Alvi and Muhammad M. Hammami. This is an open access article distributed under the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract
A simple ultraperformance liquid chromatography-tandem mass spectrometry assay for measurement of cortisol level in human saliva was developed and validated. Saliva samples containing cortisol were spiked with tolperisone as internal standard (IS) and extracted with a mixture of methyl tert-butyl ether and hexane (8:2, v:v). After solvent evaporation, residue was reconstituted in 100 μ l mobile phase. Analysis was performed on Atlantis dC18 column (2.1 × 100 mm, 3 μ m particle size) with a mobile phase composed of acetonitrile and 2 mM ammonium acetate (50:50, v:v) and delivered at a flow rate of 0.3 ml/minute. Mass spectrometry acquisition was performed with multiple reaction monitoring in positive-ion mode for cortisol and IS (m/z: 363.1 → 121.0 and 246.0 → 97.9, respectively). Retention times of cortisol and IS were about 1.35 and 2.45 minutes, respectively. The relationship between cortisol level and peak area ratio of cortisol to IS was linear in the range of 0.5-100 ng/ml. Intra- and interday coefficient of variation and bias were ≤ 9.0% and ≤12.0%, respectively. Mean extraction recoveries of cortisol and IS from saliva samples were 92% and 94%, respectively. Using the method, cortisol was found to be ≥ 86% stable in processed (24 hours at room temperature or 48 hours at -20°C) and ≥ 91% stable in unprocessed (24 hours at room temperature or 20 weeks at -20°C) saliva samples. Further, the method was successfully applied to determine daily cortisol profile in saliva samples of a healthy volunteer. 1. Introduction
Cortisol is a steroid hormone that plays an important role in regulating a wide range of physiological and pathological processes that involves immune response, electrolyte balance, blood pressure, and metabolism among others [ 1 , 2 ]. Measurement of cortisol level in biological fluids is used in the diagnosis of diseases related to adrenal, pituitary, and hypothalamic function, including Cushing’s syndrome and Addison’s disease [ 3 , 4 ].
Measurement of cortisol level in saliva samples is particularly attractive as it reflects biologically active cortisol and samples can be obtained stress-free [ 5 , 6 ]. Several studies assessed cortisol level in saliva, using radioimmunoassay (RIA) [ 7 , 8 ], enzyme immunoassay (EIA) [ 9 – 11 ], high-performance liquid chromatography (HPLC) [ 12 , 13 ], or liquid chromatography-tandem mass spectrometry (LC-MS/MS) [ 14 – 17 ]. Salivary cortisol levels measured by RIA are strongly correlated with levels measured by a highly sensitivity EIA (r = 0.98, P < 0.001) [ 11 ] and LC-MS/MS (r = 0.99, P < 0.01) [ 15 ]. However, although immunoassays have high sensitivity, they often suffer from low selectivity due to cross-reactivity with related substances [ 18 ]. On the other hand most of the reported LC-MS/MS assays are based on solid-phase extraction [ 15 , 17 ] and/or the use of deuterium-labeled cortisol as an internal standard (IS) [ 14 , 16 ], which may not be feasible in some clinical laboratories.
Here we describe a simple, precise, and rapid ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) assay for determination of cortisol level in human saliva using tolperisone as an internal standard (IS). The method was validated according to US FDA guidelines [ 19 ] and used to determine cortisol stability in saliva under various clinical laboratory conditions and applied to monitor the level of cortisol in saliva samples collected from healthy volunteers. 2. Experimental 2.1. Chemicals and Reagents
All chemicals were of analytical grade unless stated otherwise. Hydrocortisone (cortisol) and tolperisone were purchased from Acros organic, NJ, USA, and Sigma-Aldrich, MO, USA, respectively. Ammonium acetate, methyl tert-butyl ether, hexane, and acetonitrile (HPLC grade) were purchased from Fisher Scientific, NJ, USA. HPLC grade water was prepared by reverse osmosis and further purified by passing through a Synergy Water Purification System (Millipore, Bedford, MA, USA). The study was approved by the Research Ethics Committee of King Faisal Specialist Hospital & Research Centre, Riyadh, Saudi Arabia, under Research Advisory Council (RAC# 2160008). 2.2. Instrument and Chromatographic Conditions
The liquid chromatograph tandem mass spectrometer (LC-MS/MS) consists of Xevo-TQD detector equipped with Z-spray, an atmospheric pressure ionization (API) interface, Acquity UPLC H-Class system, integrated solvent, and sample manager (Waters Corporation, Milford, MA, USA). Analysis was performed at room temperature using a reversed phase Atlantis dC18 column (2.1 × 100 mm, 3 μ m particle size), steel column protected by a column guard in-line filter (2 mm, 0.2 μ m). The mobile phase was composed of 2 mM ammonium acetate and acetonitrile (50:50, v:v). It was filtered using (47 mm, 0.2 μ m pore size) Supor membrane disc filter (Pall Gelman Laboratory, MI, USA) and delivered at a flow rate of 0.3 ml/minute. The electrospray ionization (ESI) source was operated in the positive-ion mode at a capillary voltage of 1.5 kV and cone voltage of 36 V. Nitrogen was used as the nebulizing and desolvation gas at a flow rate of 1000 L/hr. Argon was used as the collision gas maintaining cell pressure at 3.6 E −003 mbar. An optimum collision energy of 20 eV was applied for both cortisol and IS. The ion source and the desolvation temperatures were maintained at 150°C and 500°C, respectively. Cortisol and IS were detected and quantified in the positive-ion mode; product ion response was measured in multireaction monitoring (MRM) mode at set transitions mass to charge (m/z) of 363.1 → 121.0 and 246.0 → 97.9, respectively. Mass lynx Ver 4.1 (Waters Corporation, Milford, MA, USA) software working under Microsoft Window XP professional environment was used to control the instrument parameters, data acquisition, peak integration, peak smoothing, and signal-to-noise ratio measurements. 2.3. Preparation of Standard and Control Samples
Cortisol and IS stock solutions were prepared in methanol (1.0 μ g/ml). Nine calibration standards in the range of 0.5-100 ng/ml and four quality control concentrations (0.5, 1.5, 50, and 90 ng/ml) were prepared in normal human saliva. IS working solution (20 ng/ml) was prepared in water. Aliquots (1.0 ml) of saliva samples were transferred into 7 ml glass culture tubes and stored at -20°C until used. 2.4. Saliva Samples for Calibration Standards and Controls
Unstimulated saliva samples were collected from healthy volunteers by direct spitting in sterile (110 mm × 28 mm dia) Corning 50 ml centrifuge tubes (Sigma-Aldrich, USA) and stored at -20°C until analyzed. 2.5. Preparation of Samples
50 μ l of the IS working solution (20 ng/ml) was added to each 1.0 ml unknown saliva sample, calibration standard, or quality control samples in 7 mL glass culture tubes and vortexed for 30 seconds. 4.0 ml mixture of methyl tert-butyl ether and hexane (8:2, v:v) was added, vortexed for two minutes, and centrifuged at 4000 rpm for 15 minutes at 20°C. The supernatant clear layer was transferred to a clean borosilicate culture tube and dried under gentle steam of nitrogen at 40°C. The residue was reconstituted in 100 μ l mobile phase, 2 mM ammonium acetate and acetonitrile (50:50, v:v) and 10 μ l of the clear solution was injected into the LC-MS/MS system. 2.6. Extraction Recovery
Extraction recovery of cortisol was measured by comparing cortisol peak areas in two samples. In one sample blank saliva was spiked with cortisol and then extracted. In the second sample, blank saliva was extracted first and then spiked with the same amount of cortisol. This was done in four replicates at four concentrations (0.5, 1.5, 50, and 90 ng/ml). Recovery of the IS was determined in the same fashion at a concentration of 20 ng/ml. Extraction recovery was calculated as mean cortisol (or IS) peak area in spiked-before-extraction samples divided by mean cortisol (or IS) peak area in spiked-after-extraction times 100. 2.7. Stability Studies
Two QC samples concentrations (1.5 and 90 ng/ml) in saliva were used for stability studies. Five aliquots of each concentration were extracted and immediately analyzed (baseline). Five aliquots of each concentration were allowed to stand on the bench-top for 24 hours at room temperature; five aliquots were stored at -20°C for 20 weeks, before being processed and analyzed; and five aliquots were processed and stored at room temperature for 24 hours or at -20°C for 48 hours before analysis. Fifteen aliquots of each concentration were stored at −20°C for 24 hours. They were then left to completely thaw unassisted at room temperature. Five aliquots were analyzed and the rest stored at to -20°C for another 24 hours. The cycle was repeated three times. 2.8. Calculations
In order to correct for endogenous level of cortisol in “blank” saliva, we used the difference in peak area ratios between each consecutive concentration as the response (rather than the peak area ratio) [ 20 ]. The difference in peak area ratio was plotted against the concentration. Bias (%) was calculated as the difference between measured and nominal concentration divided by nominal concentration times 100, whereas coefficient of variation (%) was calculated as standard deviation divided by mean concentration times 100. 2.9. Matrix Effect
Matrix effect was evaluated by comparing the peak areas of extracted blank saliva that was then spiked with cortisol at four concentrations (0.5, 1.5, 50, and 90 ng/ml) and IS (20 ng/ml) with the corresponding peak areas obtained by direct injection of standard solutions prepared in mobile phase. 2.10. Method Validation
The method was validated (specificity, recovery, linearity, accuracy, precision, and stability) according to the US Food and Drug Administration (FDA) bioanalytical method validation guidance [ 19 ]. 2.11. Saliva Samples from a Healthy Volunteer
About 4.0 ml saliva samples were collected within 20-30 minutes by direct spitting in sterile Corning 50 ml centrifuge tubes. Samples were collected starting at 4:30, 10:00, 15:30, 19:30, and 22:00 hours on a rest day and on a work day and stored at -20°C until analyzed. 3. Results 3.1. Separation and Quantification
Figure 1 depicts the chemical structures of cortisol and tolperisone (IS). Liquid chromatographic (LC) conditions were optimized using a mobile phase composed of 2 mM ammonium acetate and acetonitrile (50:50, v:v) at a flow rate 0.3 ml/minute. The relatively high proportion of acetonitrile facilitated column low back pressure and shorter run time (< 3.0 minutes). The product and precursor ions were determined by infusing a standard mixture of cortisol and tolperisone (1.0 μ g/ml in methanol) in the mass spectrometer using a configured software program (IntelliStart, obtained from Waters Corporation, Milford, MA, USA). Cortisol and IS each produced two product ions peaks: 363.1 → 121.0 or 363.1 → 97 and 246.0 → 97.9 or 246.0 → 69.9, respectively. Transition 363.1 → 121.0 for cortisol and transition 246.0 → 97.9 for IS were chosen to quantitate cortisol level, since they gave the best response. Figure 2 depicts total ion current (TIC) and MRM chromatograms of cortisol and IS. Figure 1: Chemical structures of cortisol and tolperisone (IS). Figure 2: Total ion current and multiple reaction monitoring chromatograms of cortisol and the internal standard (IS) tolperisone in human saliva. 3.2. Matrix Effect
Matrix effect is common in atmospheric pressure ionization LC-MS/MS analysis. It is mainly due to interference of molecules originating from the sample matrix that coelute with the compound(s) of interest during the process of ionization, causing ionization suppression or enhancement. Matrix effect, usually up to ±15%, is considered as insignificant [ 21 , 22 ]. Mean matrix effect was an ion suppression of -9.7% and -13.7% for cortisol and IS, respectively. 3.3. Specificity
The specificity of the assay was determined by screening six different batches of blank human saliva, in addition to seven cortisol-related compounds (cortisone, progesterone, 17 α – hydroxy progesterone, prednisone, prednisolone, methyl prednisone, and testosterone). All solutions were 1.0 μ g/ml in methanol: water (1:1, v:v) and 10 μ l was injected into the system. No interference with the peaks of cortisol or the IS was obtained. Figure 3 depicts representative chromatograms of human saliva that was used in preparation of calibration curve and quality control samples. Figure 3: Multiple reaction monitoring chromatogram of blank human saliva used in preparation of standard and quality control sample. (a) Blank saliva, (b) blank saliva spiked with internal standard. 3.4. Recovery
The results of extraction recovery of cortisol and the IS are presented in Table 1 . Mean extraction recoveries were 92% for cortisol and 94% for the IS. Table 1: Extraction recovery of cortisol and internal standard (IS) from saliva. 3.5. Linearity and Limit of Detection and Quantification
Linearity of the assay was evaluated by analyzing a series of cortisol standards at nine different concentrations over the range of 0.5–100 ng/ml in saliva. Corresponding peak area ratios and concentrations were subjected to regression analysis. Mean equation obtained from eight standard curves was y= 0.0188 (x) + 0.0117, with R 2 (SD) = 0.9960 (0.0039). The detection and quantification limits were as 0.3 ng/ml and 0.5 ng/ml, respectively. Figure 4 represents UPLC-MS/MS chromatograms of four QC samples (0.5, 1.5, 50, and 90 ng/ml) spiked with 20 ng/ml of IS. Figure 4: Multiple reaction monitoring chromatograms of four quality control samples ((a) 0.5, (b) 1.5, (c) 50, and (d) 90 ng/ml) spiked with 20 ng/ml internal standard (IS). 3.6. Precision and Bias
The intraday and interday precision and bias (Table 2 ) were evaluated by analyzing four QC samples (0.5, 1.5, 50, and 90 ng/ml). The intraday precision and bias (n = 10) ranged from 2.4% to 9.0% and from –5.5% to 12.0%, respectively. The interday precision and bias were determined over three different days (n=20). They ranged from 3.9% to 8.4% and from –2.0% to 10.3%, respectively. Table 2: Intra-and inter-run precision and accuracy of cortisol assay. 3.7. Stability
Cortisol and IS stability in processed and unprocessed saliva samples was investigated (Table 3 ). Cortisol in processed samples was found to be stable for 24 hours at room temperature (≥88%) and 48 hours at −20°C (86%). Cortisol in unprocessed samples was stable for at least 24 hours at room temperature (≥92%), 20 weeks at −20°C (≥91%), and after three freeze-thaw cycles (≥93%). Further, no significant change in chromatographic behavior of cortisol or the IS was observed under any of the above conditions. Table 3: Stability of cortisol in human saliva. 3.8. Application of the Method
The method was used to determine salivary cortisol profile in a healthy volunteer on two different days. The results are presented in Figure 5 . As expected, cortisol levels were highest in the early morning period and declined to unmeasurable level at 10:00 to 10:30 PM. Figure 5: Level of cortisol in saliva samples collected from a healthy volunteer at different intervals on two different days. 4. Discussion
Measurement of cortisol level plays an important role in the diagnosis of adrenal dysfunction as well as in studying related physiological conditions [ 1 – 4 ]. A number of liquid chromatographic methods have been reported for cortisol determination in various biological matrixes [ 23 , 24 ]. Measuring cortisol level in saliva has several advantages over other matrixes, including convenience in sample collection, avoidance of stress associated with vein puncture, and measuring biologically active cortisol level rather than total cortisol level [ 5 ].
Few LC-MS/MS-based assays have been reported for cortisol measurement in saliva [ 14 – 17 ]; they used solid-phase extraction and/or deuterium-labeled cortisol as IS. In general deuterium-labeled analytes have been used as IS in LC-MS/MS analysis, since they permit identical fragmentation of the IS and the assayed analyte. However, advancement in LC-MS/MS softwares allowed the successful use of non-deuterium-labeled ISs. We elected to use a chemically similar compound to cortisol, tolperisone, as an IS. Tolperisone gave major fragment peak response at collision energy of 16 eV compared to 20 eV for cortisol. Nevertheless, collision energy of 20 eV produced reliable and consistent results for the IS and thus was chosen.
We used a simple liquid-liquid extraction for sample preparation. Liquid-liquid extraction is considered a classic approach and is widely used in sample preparation for qualitative and quantitative analysis. Its main advantage is low cost, as it does not require expensive equipment compared to solid-phase extraction.
Cortisol level in saliva ranges from 0.6 to 15 ng/ml (1.5-40 nmol/L) when measured by immunoassay [ 7 ]. Although immunoassays are highly sensitive, they often suffer from cross-reactivity with cortisol-related compounds. In fact, cortisol levels obtained by immunoassays are 2.7 times higher than those obtained by LC-MS/MS [ 16 ]. Using the current assay, we found that cortisol level was about 3.5 ng/ml in the early morning and declined to unmeasurable level in the evening, in agreement with previous report [ 25 ]. 5. Conclusion
The described UPLC-MS/MS method is simple, precise, and accurate for rapid measurement of cortisol level using 1.0 ml human saliva. The method uses readily available internal standard and was successfully used to determine cortisol stability under various laboratory conditions. Further, it was successfully applied to determine cortisol level in saliva samples obtained from a healthy volunteer. Data Availability
The data used to support the findings of this study are available from the corresponding author upon request. Conflicts of Interest
The authors declare that they have no conflicts of interest. Authors’ Contributions
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Kirschbaum, “Comparison of salivary cortisol as measured by different immunoassays and tandem mass spectrometry,” Psychoneuroendocrinology , vol. 38, no. 1, pp. 50–57, 2013. View at Publisher · View at Google Scholar · View at Scopus B. A. Jonsson, B. Malmberg, A. Amilon, G. A. Helene, and P. Orbaek, “Determination of cortisol in human saliva using liquid chromatography–electrospray tandem mass spectrometry,” Journal of Chromatography B Analyt Technol Biomed Life Sci , vol. 784, no. 1, pp. 63–68, 2003 (Arabic). View at Publisher · View at Google Scholar H. Kataoka, E. Matsuura, and K. Mitani, “Determination of cortisol in human saliva by automated in-tube solid-phase microextraction coupled with liquid chromatography–mass spectrometry,” Journal of Pharmaceutical and Biomedical Analysis , vol. 44, no. 1, pp. 160–165, 2007. View at Publisher · View at Google Scholar I. Perogamvros, L. J. Owen, B. G. Keevil, G. Brabant, and P. J. Trainer, “Measurement of salivary cortisol with liquid chromatography-tandem mass spectrometry in patients undergoing dynamic endocrine testing,” Clinical Endocrinology , vol. 72, no. 1, pp. 17–21, 2010. View at Publisher · View at Google Scholar · View at Scopus “Bioanalytical Method Validation,” Guidance for Industry US Department of Health Services, Food and Drug Administration, CDER, CVM, 2018. S. N. Alvi, S. Al-Dgither, and M. M. Hammami, “Development and validation of LC-MS/MS method for determination of testosterone level in human saliva using lovastatin as internal standard,” Journal of Bioequivalence & Bioavailability , vol. 5, no. 6, pp. 228–232, 2013. View at Publisher · View at Google Scholar B. K. Matuszewski, M. L. Constanzer, and C. M. Chavez-Eng, “Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS,” Analytical Chemistry , vol. 75, no. 13, pp. 3019–3030, 2003. View at Publisher · View at Google Scholar R. Dams, M. A. Huestis, W. E. Lambert, and C. M. Murphy, “Matrix effect in bio-analysis of illicit drugs with LC-MS/MS: influence of ionization type, sample preparation, and biofluid,” Journal of The American Society for Mass Spectrometry , vol. 14, no. 11, pp. 1290–1294, 2003. View at Publisher · View at Google Scholar · View at Scopus S. N. Alvi and M. M. Hammami, “Measurement of cortisol in human plasma and urine by ultra performance liquid chromatography-tandem mass spectrometry,” Asian Journal of Pharmaceutical and Clinical Research , vol. 11, no. 6, pp. 199–203, 2018. View at Publisher · View at Google Scholar · View at Scopus U. Turpeinen and E. Hämäläinen, “Determination of cortisol in serum, saliva and urine,” Best Practice & Research Clinical Endocrinology & Metabolism , vol. 27, no. 6, pp. 795–801, 2013. View at Publisher · View at Google Scholar · View at Scopus C. Kirschbaum and D. H. Hellhammer, “Salivary Cortisol,” in Encyclopedia of Stress , vol. 3, pp. 379–383, 2000. 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<h1>Viceland’s ‘Rise’ Relives the Dakota Access Pipeline Protests</h1>

Viceland’s ‘Rise’ Relives the Dakota Access Pipeline Protests

Coming just after President Trump signed an order to approve the pipeline, this series about indigenous resistance movements makes for melancholic viewing.

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<h1>Spurfy’s Fasting Log</h1>

Spurfy’s Fasting Log

After careful consideration I’ve decided that I’m going to need to taper off of narcotic pain meds and prednisone/oral hydrocortisone before I start full-on fasting. I’m at the point where serious adrenal suppression has probably occurred and stopping the corticosteroids cold-turkey just isn’t an option without considerable risk.
As many of you probably know, cortisol elevates during fasting but not if negative feedback inhibition from exogenous corticosteroids has shut down the adrenal glands. This is no different than the HPG-axis suppression from exogenous androgens, except that one actually needs cortisol to live, while testosterone isn’t needed to sustain life.
Also, glucocorticoids elevate both glucose and insulin, and ketosis will suffer greatly if I attempt to fast while I’m tapering.
The opioids I will also be tapering off of and if withdrawal is or becomes an issue I have gabapentin, baclofen, and diazepam to lessen the discomfort and to make the transition smoother.
Starting today until March 20, I’ll be consuming a single small ketogenic meal after sunset with no water during the day. For the first few days I’ll probably be taking 120-180 mg of pseudoephedrine upon waking to help control my appetite, as both opioids and corticosteroids make me constantly hungry.
Starting March 21, at the conclusion of my drug tapers, I will begin fasting with only water, lime juice, electrolytes, black coffee or green tea (if needed), oral hydrocortisone in the morning prior to any anticipated heavy exertion (if any), tianeptine (if needed for mood), a nicotine patch if my energy really starts crashing or the pain from my discs or osteoarthritis becomes too overwhelming, and melatonin at bedtime.
For exercise I will be walking daily once the actual fasting starts. There will likely be no before and after pics, and no daily weigh-in. I’m doing this as a gift to my son, who is 4, who needs me to live a very long and healthy life. All previous fasts have had a large aesthetic element but I want this one to be completely selfless. I’ll log my weight once per week.
I will not read or watch any news, nor any TV or movies except uplifting documentaries, and the only social media I will be active in is Instagram (pics of my son) and this forum.
At the conclusion (which right now is open-ended) I hope to have healed my body enough that I can begin training again, and most-importantly to try to see life as a gift instead of an affliction. Presently, the only time I’m not abjectly miserable is the short period each week I get with my son. It would be nice to be able to see at least some of the joy and wonder and amazement of this world like my son does, instead of seething with sadness, anger, resentment, and regret. I can only barely remember what it felt like to see my life as anything but a tremendous burden. Except for when I’m with my son? all of my hope, optimism, inspiration, humor, joy, gratitude, compassion, empathy, and warmth are just gone and no matter how deeply I dig, I just can’t find them. I’m constantly filled with emptiness so profound that I have stopped loving everyone but my son and have basically abandoned all of my friendships. I used to want to change the world for the better — now I find myself hoping for a nuclear war or Yellowstone eruption. I don’t want this any more.
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<h1>How This Ardmore Fitness Entrepreneur Came to Terms With Her Chronic Disease</h1>

How This Ardmore Fitness Entrepreneur Came to Terms With Her Chronic Disease

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